Treatment optimization of maintenance immunosuppressive agents in pediatric renal transplant recipients

Abstract

Introduction:

Graft survival in pediatric kidney transplant patients has increased significantly within the last three decades, correlating with the discovery and utilization of new immunosuppressants as well as improvements in patient care. Despite these developments in graft survival for patients, there is still improvement needed, particularly in long-term care in pediatric patients receiving grafts from deceased donor patients. Maintenance immunosuppressive therapies have narrow therapeutic indices and are associated with high inter-individual and intra-individual variability.

Areas covered:

In this review, we examine the impact of pharmacokinetic variability on renal transplantation and its association with age, genetic polymorphisms, drug-drug interactions, drug-disease interactions, renal insufficiency, route of administration, and branded versus generic drug formulation. Pharmacodynamics are outlined in terms of the mechanism of action for each immunosuppressant, potential adverse effects, and the utility of pharmacodynamic biomarkers.

Expert opinion:

Acquiring abetter quantitative understanding of immunosuppressant pharmacokinetics and pharmacodynamic components should help clinicians implement treatment regimens to maintain the balance between therapeutic efficacy and drug-related toxicity.

1. Introduction

Kidney transplantation is the treatment of choice for pediatric patients with end-stage renal disease (ESRD). Graft survival, however, currently requires long-term immunosuppression to prevent graft rejection. Immunosuppressive therapy in kidney transplant patients has evolved over the last five and a half decades. As more effective immunosuppressants have become available, patient outcomes have improved, with less acute graft rejection within the first 12 months, and increased graft survival at five years after renal transplantation [1–3]. These improvements are attributed in part to the development of more effective immunosuppressant drugs [1,4,5]. Improved immunosuppression contributes to extended host and graft survival in pediatric kidney recipients. Therefore, a clear understanding of the standard of care immunosuppressants is essential.

1.1. Immunosuppressant use in pediatric renal transplantation

The use of immunosuppressants in children undergoing renal transplantation consists of induction and maintenance. The objective of induction is to prevent early acute graft rejection in these high-risk patients. Patients are generally treated with high-dose immunosuppressants during the first few months after transplantation when the risk of acute rejection is the highest. In the United States, the currently utilized regimens include corticosteroids (e.g. prednisone) as well as T-lymphocyte depleting agents (e.g. polyclonal anti-thymocyte globulin and monoclonal lymphocyte-depleting antibodies) and monoclonal antibodies targeting the IL-2 receptor (e.g. basiliximab) [6,7]. Maintenance immunosuppressants, as single drug or, more commonly in combination, are used to attenuate transplant recipients’ immune response in long-term treatment schedules and most often include combinations of tacrolimus or cyclosporine with mycophenolate or azathioprine with or without prednisone.

1.2. Development of immunosuppressant regimens

Improvements to immunosuppressive maintenance therapy have advanced over the last several decades. Several combination maintenance therapies are commonly used in pediatric kidney patients with functioning grafts [3]. These combinations generally included a steroid, a calcineurin inhibitor (CNI), and an anti-proliferative agent. In 2019, the most common initial maintenance immunosuppression regimen was tacrolimus, mycophenolate, and a steroid [8]. Figure 1 displays the percentage of patients treated with the most popular regimens for 1996–2001 in panel A, 2002–2007 in panel B, and 2008–2013 in panel C. The use of cyclosporine has decreased markedly over the years, with over 60% of patients in 1996 treated with a regimen that included cyclosporine one year after allograft surgery, to less than 3% of patients in 2013. The ‘other’ category mainly includes regimens with newer mammalian targets of rapamycin (mTOR) inhibitors, such as sirolimus and everolimus, and monoclonal antibodies, such as basiliximab., As described by Hart et al. [2] these trends continued through 2015 as tacrolimus and mycophenolate were utilized in initial maintenance immunosuppression in over 96% and 93% of reported transplants, respectively, while mTOR inhibitors were used ~8% by one-year posttransplant, and the use of corticosteroids decreased by ~4% at one-year posttransplant from 2014 and 2015. These combinations may continue to change as clinical experience with these agents in pediatric patients is disseminated [9].

Figure 1.

Timeline of significant developments in immunosuppressive therapy and initial US approvals of therapies beginning in 1955 through 2011 [54].

By the 2000s, organ survival at one year following transplantation in pediatric patients had increased from about 81% for deceased donors and ~91% for living donors to over 96% in 2015 [1,2]. Longer-term graft survival at five years after transplantation has improved as well. In the early 1990s, ~62% and ~75% of deceased and live donor grafts survived; in 2015, ~82% of deceased donor graft recipients and almost ~89% of living donor grafts survived [1,2]. Graft survival and a brief listing of significant developments in kidney transplant treatments are shown in Figures 2 and 3, respectively. While graft survival for pediatric patients has improved, improvement is needed in long-term graft outcomes. Pape recently suggested that stratification strategies, based on patient characteristics may improve outcomes based on a review of pediatric transplant clinical trial data [10].

Figure 2.

Immunosuppressive therapy from the time of transplant in kidney transplant, Panel A Transplant era (1996 to 2001), Panel B Transplant era (2002–2007), Panel C Transplant era (2008 to 2013) [1].

Figure 3.

Deceased and living donor graft survival at 1-year post-transplant (1991 through 2015) [3,2].

1.3. Immunosuppression

Understanding the factors influencing appropriate immunosuppression in pediatrics is critical to extending host and graft survival. These factors include pharmacokinetics (PK), pharmacodynamics (PD), and pharmacogenomics considerations. This review presents a history of maintenance immunosuppression in pediatric renal transplant patients and discusses several factors for clinicians to consider in treatment plans.

2. Monitoring of pediatric transplant recipients

During the post-transplant period, clinical monitoring of the transplanted kidney is utilized widely to detect any potential allograft dysfunction. Serum creatinine levels are the most common approach to monitor kidney function [11]; however, the ‘gold standard’ for checking potential kidney injury via immunologic processes is renal allograft biopsy [12]. Children are unique as they often receive an adult-sized kidney from a parent, which may undergo considerable deterioration before serum creatinine rises. Therefore, serum creatinine may be a poor indicator of allograft dysfunction in children. Relying on an increase in serum creatinine as an indication to perform renal allograft biopsy may result in finding irreversible allograft damage. Protocolized renal allograft biopsy rather than biopsy ‘for cause’ may demonstrate rejection even before a rise in serum creatinine levels is observed, particularly in small children [13].

3. Long term considerations for pediatric transplant recipients

Continued development of pediatric renal transplant recipients requires careful attention to long-term outcomes beyond the short-term survival of the transplanted kidney. These include effects on pubertal maturation and fertility, metabolic problems, and the development of mineral and bone disorders resulting in bone pain, fractures, growth failure, and ectopic calcifications.

3.1. Cardiovascular risk

After kidney transplantation, children have a high prevalence of many cardiovascular risk factors, including obesity, hypertension, dyslipidemia, and insulin resistance. While all these factors negatively affect graft function, they also correlate with accelerated cardiovascular diseases, including ischemic heart disease and premature dilated cardiomyopathy [14]. There is evidence that the use of some immunosuppressants may increase the risk of metabolic syndrome [15]. While frequent at the time of transplantation, the prevalence of metabolic syndrome significantly increases one year after kidney transplantation [16]. As metabolic syndrome is identified as a significant risk factor for cardiovascular diseases [17], children after kidney -transplantation should be considered at high risk for metabolic abnormalities and receive appropriate follow-up [17].

3.2. Post-transplant lymphoproliferative disorder

Another problem deriving from organ transplantation is post-transplant lymphoproliferative disorder (PTLD), which may occur during immunosuppressive therapy and is more common in pediatric patients than in adult patients[18]. In adult renal recipients, PTLD has been reported to occur in 1.2% of the cases, while in children, it occurs in around 10% of kidney transplants. Primary infection with the Epstein-Barr virus increases the risk of developing PTLD over 6-fold [19]. Therefore, the higher incidence of PTLD in pediatrics could be related to the higher frequency of recipients who are initially seronegative for Epstein-Barr virus.

3.3. Cytomegalovirus infection

Human cytomegalovirus (hCMV) infection is an additional post-transplant complication in pediatric kidney transplant recipients. Prior to the introduction of prophylactic measures, ~15% of pediatric kidney recipients developed hCMV end-organ disease [20]. The incidence of hCMV disease has decreased with the introduction of hCMV prophylaxis [20]. Kranz et al. have reported long-term outcomes in renal transplant pediatric recipients with CMV infections [21]. The authors demonstrated that hCMV infection occurred in ~20% of children, and ~10% of children developed hCMV end-organ disease. Children receiving organ transplant have an elevated risk of acquiring primary hCMV infection since, unlike adults, a higher percentage of children are seronegative prior to transplantation [20]. hCMV seronegative and seropositive recipients have an increased risk of hCMV infection when receiving kidney transplants from hCMV positive donors [21,22]. The acute kidney rejections during or after hCMV infection/disease were positively associated with seropositivity of the kidney donor regardless of hCMV-positivity of the recipient [21]. Monitoring hCMV status in pediatric kidney transplant recipients is an essential part of strategy planning against kidney rejection and hCMV end-organ disease development.

There are three prophylaxis strategies to manage hCMV disease development: anti-CMV prophylaxis, preemptive therapy, and hybrid therapy [23]. Preemptive therapy consists of monitoring hCMV levels and initiation of an anti-viral treatment when a positive threshold of hCMV is reached. Prophylactic treatment involves the treatment of all patients except seronegative recipients of a seronegative kidney with anti-CMV therapy [23]. Hybrid therapy consists of initial prophylactic anti-hCMV therapy followed by hCMV level monitoring [20,22]. As a part of prophylactic therapy, pediatric patients can be treated with intravenous ganciclovir or oral valganciclovir [20,22]. The Transplantation Society International CMV Consensus Group recommends 2–4 weeks intravenous ganciclovir/valganciclovir followed by hCMV monitoring for hCMV positive recipients and 2–4 weeks intravenous ganciclovir/valganciclovir followed by monitoring or 3–6 months of intravenous ganciclovir/valganciclovir, similar to recommendations in adults [20,22]. There is some in vitro evidence suggesting that mTOR inhibitors may have anti-CMV effects [22]. There is strong evidence suggesting that hCMV seropositive kidney recipients receiving mTOR inhibitors have a lower incidence of hCMV infection or disease [22]. Based on these studies, the Transplantation Society International CMV Consensus Group has recommended the use of mTOR inhibitors as a potential approach in seropositive recipients of a kidney transplant [22]. It is unclear whether mTOR inhibitor treatment will prevent hCMV infection when a donor is hCMV seropositive [22]. Therefore, the CMV prophylaxis strategy may be influenced by the hCMV status of the recipient, hCMV status of the transplanted kidney, and overall immunosuppression strategy.

3.4. Considerations associated with pediatric growth and development

Changes in bone and mineral metabolism have been associated with the last stages of chronic kidney disease. Thus, although they are already present at the time of kidney transplantation, bone loss often advances after kidney transplantation, with three main contributing factors: preexisting renal osteodystrophy at the time of transplantation, glucocorticoid treatment, and long-term graft function. High bone turnover is managed by parathyroid hormone (PTH) suppression via vitamin D analogs and calcimimetics., Low bone turnover is managed by avoiding vitamin D and reduced doses of calcium [24]. Consequently, appropriate mineral management during the post-transplant period is indispensable to avoid or reduce potential abnormalities [25].

Long-term steroid exposure after renal transplantation can delay puberty in pediatric renal transplant patients [26]. Growth can also be severely affected in children undergoing renal transplantation. Growth after transplantation is influenced by age at transplantation [27], allograft function, and steroid exposure [28]. Although growth after transplantation is usually satisfactory, most individuals’ final height is less than their expected height. This is associated with short stature, especially in adolescence and also in adulthood, which can significantly impact self-esteem and quality of life; it can be crucial to address [29].

Glucocorticoids have a well-known growth-suppressing effect [30]. Therefore, the use of steroids/glucocorticoids in pediatric kidney transplantation is usually avoided or minimized [31,32]. A few reports and clinical studies have evaluated the withdrawal or avoidance of steroids in pediatric patients [33–42]. A report by Grenda et al. l [42]. assessed the effect of two regimens (tacrolimus, mycophenolate, and steroids or tacrolimus, mycophenolate, and daclizumab) on height standard deviation score, as well as the safety and efficacy of the regimens during the first six months after renal allograft. Their findings indicated that early steroid withdrawal significantly benefited growth in prepubertal children without significant increases in graft rejection or loss in the short-term. However, it should be noted that this steroid reduction strategy may be associated with glomerulonephritis [43]. The study of steroid withdrawal or avoidance in pediatric patients should be advanced, particularly during longer follow-up times.

Drug-metabolizing enzymes change significantly throughout childhood. Indeed, cytochrome P450 (CYP), CYP3A expression, the main pathway of tacrolimus metabolism, is low at birth and increases until 6–12 months of age, when it reaches 50% of adult expression [44]. Ferraresso and colleagues have demonstrated that CYP3A4 expression varies significantly with increasing pediatric age and influences tacrolimus dosing [45]. These ontogeny-related changes may influence immunosuppressive drug disposition and are essential considerations for immunosuppressive therapies in pediatric patients.

As a chronic disease, post-transplant treatment adherence is essential to maintaining graft function and quality of life. About 30% of pediatric kidney transplant patients do not follow the immunosuppressive regimen correctly, being more pronounced in adolescents than in young children [46].

4. Pediatric pharmacology of immunosuppressants

The pharmacology of the most commonly utilized maintenance immunosuppressants in pediatric kidney transplantation must be considered in a treatment plan. These include CNIs (tacrolimus and cyclosporine), mTORs (sirolimus), inosine monophosphate dehydrogenase inhibitors (mycophenolic acid, often in prodrug mycophenolate mofetil (MMF, trade name CellCept)). Pharmacology of other immunosuppressants, including azathioprine (purine antagonist), everolimus (mTOR), and glucocorticoids as well as IL-2 receptor antagonists (basiliximab), are discussed in other literature [47–51].

4.1. Pharmacokinetics (PK)

Understanding the PK (how the body affects a drug) of specific immunosuppressants is critical to appropriate dosing and monitoring. PK data in the pediatric transplant population is limited, and often dosing is established using data from adults. Developmental differences in the ontogeny of drug-metabolizing enzymes and elimination pathways influence the success of renal transplantation in children. In pediatric transplant patients, immunosuppressants must be selected and dosed carefully due to altered metabolism, poor absorption, frequent drug-associated adverse reactions, and apparent rapid clearance [52]. This section reviews the PK of tacrolimus, cyclosporine, sirolimus, and mycophenolic acid (MPA). We use MPA as the active compound of the pre-systemically hydrolyzed prodrug mycophenolate mofetil (MMF, CellCept) and the enteric-coated mycophenolate sodium (EC-MPS, Myfortic) found in delayed-release formulations.

4.1.1. Absorption

Cyclosporine has a highly variable oral availability dependent on formulation and transplant type, ranging from <10% and up to 89%, with greater availability observed in kidney transplant patients [53,54]. Sirolimus and tacrolimus have low oral availability (<30%) [55,56]. MMF has high oral availability, undergoing extensive hydrolysis to mycophenolic acid (MPA), with >90% of absorption [57]. Cyclosporine, sirolimus, tacrolimus, and MPA are all substrates of ABCB1 (ATP-binding cassette sub-family C member 1), a transmembrane protein efflux transporter, also known as p-glycoprotein, affecting drug uptake from the intestinal lumen into the epithelium and associated with multidrug resistance [58]. All four immunosuppressive agents have relatively rapid absorption. The time to maximum concentration (T_max) occurs 1–2 hours post-dose [53,55–57]. The rate constant of absorption (ka) is generally derived from adults, as several samples in the absorption phase are required to derive this parameter, and the opportunity for sampling is often limited in pediatrics. An important pediatric consideration for the youngest patients is that they are likely to be administered an oral solution which is difficult to compound consistently due to variation in capsules, tablets, or powder preparations. Therefore, oral solutions may have a different absorption profile than standard oral capsules or tablets, which are often administered to older children and adults. MPA also undergoes enterohepatic recycling, as evidenced by a second peak in plasma concentrations 6–8 hours after drug administration. Enterohepatic recycling accounts for a maximum of 60% of total MPA exposure [59].

4.1.2. Distribution

All four immunosuppressants are protein-bound, but to varying degrees: cyclosporine (>90%), MPA (82%−97%) [60], sirolimus (92%) [61] and tacrolimus (72%) [62]. Cyclosporine is bound to plasma lipoproteins [63], MPA is primarily bound to albumin [60], sirolimus is also bound to lipoproteins (40%) [64], while tacrolimus is bound to both albumin and alpha-1-acid glycoprotein equally [62]. Protein binding differences in the pediatric population have not been fully established. Cyclosporine, sirolimus, and tacrolimus have an increased distribution, particularly bound to red blood cells, with blood to plasma ratios of 1.8 [63], 30.9 (CV 48.5%) [65], and 55.5 (CV 48.3%)[66], respectively. MPA is mainly distributed to the plasma, with only 0.01% in red blood cells [67].

4.1.3. Metabolism

Cyclosporine, sirolimus, and tacrolimus are extensively metabolized by CYP3A4/5, forming inactive metabolites [53,55,56,68]. It should be noted that the primary route of elimination for cyclosporine, sirolimus, and tacrolimus is metabolism. MPA undergoes glucuronidation by uridine 5ʹ-diphospho-glucuronosyltransferase (UGT) including UGT1A8, 1A9, and 2B7 [69] to form several glucuronide metabolites. The acyl-glucuronide metabolite is the primary metabolite and, although it is pharmacologically inactive, plays an essential role in enterohepatic recycling [59].

4.1.4. Elimination

Cyclosporine, sirolimus, and tacrolimus are mainly excreted in the bile and eliminated in the feces, whereas MPA is primarily eliminated in the urine. As occurs with transplantation of other organs, the clearance per kilogram is higher in younger pediatric renal transplant patients than adults, resulting in a shorter half-life compared to older children/adults [70–73]. Therefore, these patients require a shorter dosing interval or a higher dosage of immunosuppressant (per unit of body surface area or body weight) to achieve similar concentrations or exposure to older children/adults [74]. The difference in clearance between neonates and children/adults for cyclosporine, sirolimus, and tacrolimus is related to CYP3A, ontogeny. While CYP3A5 may show only moderate changes during the first year of life [75], CYP3A4 expression is minimal in a fetus (around 30% of adult activity) [76], and the expression of CYP3A7 is dominant compared to adults [44,77,78]. CYP3A4 increases to 50% in adults by one year of age and peaks in early childhood before normalizing to adult levels in adolescence [44]. The differences in MPA disposition might be due to maturation in UGT1A9 and UGT2B7, but it needs further investigation [44,79]. Therefore, the patient’s age at the time of transplantation is an important consideration when selecting the dose of an immunosuppressant.

4.1.5. Factors that influence immunosuppressant pharmacokinetics

Pharmacokinetics can be influenced by several factors, including demographic characteristics (e.g. age and body weight), pathophysiological biomarkers (e.g. serum creatinine, liver enzymes, and albumin), and pharmacogenetics. These influences are determined from large PK trials and increasingly through population PK modeling. In population modeling, the factors that influence PK are called covariates, which explain the inter-patient variability. These covariates, along with the model, can be used to predict the drug’s PK behavior. Table 1 provides a summary of significant covariates determined from population PK models in pediatric renal transplant patients.

Table X.

Examples of pharmacogenomic considerations

Immunosuppressant	Gene	Variant or Effector	Effect
Cyclosporine	SXR [1-3]	g.-205–200 delGAGAAG	↑ CsA exposure
		↑ CYP3A4/5	↑ SXR expression
		Corticosteroids	↑ SXR expression
	ABCB1 [3-5]	c.1236C>T,  c.2677G>T	↓ Weight-adjusted CsA dose with age
		C3435T and g2677	↑ CsA exposure
Tacrolimus	CYP3A5 [6-16]	CYP3A5*3/*3	↑ Tac Cmin, Cmax, and AUC0–24
MPA	UGT1A9 [17,18]	UGT1A9 –331	↑ MPA exposure
	UGT2B7 [17,18] [19,20]	UGT2B7 –900 SNP	↑ MPA exposure
	MRP	MRP2–24T>C and UGT1A9–440C>T	↑ MPA exposure
Sirolimus	CYP3A5 [17,21,22]	CYP3A5*1	↓ Sirolimus AUC/dose, Cmax, and Cmin

SXR – xenobiotics receptor; CYP – cytochrome P450; UGT – UDP -glucuronosyltransferase; MRP2 – Multidrug resistance-associated protein 2; POR – Cytochrome P450 oxidoreductases; CsA – Cyclosporine; Tac – Tacrolimus; MPA – Mycophenolate mofetil

4.2. Pharmacodynamics

Cyclosporine, tacrolimus, mycophenolate mofetil, and sirolimus have unique mechanisms of action (Figure 4) and are associated with potentially adverse effects (AE). Clinically meaningful PD biomarkers are essential for directing treatment and improving outcomes.

Figure 4.

Mechanism of action for calcineurin inhibitors (cyclosporine, tacrolimus), Inosine-monophosphate dehydrogenase inhibitors (mycophenolic acid), and mTOR inhibitors (sirolimus and everolimus); MPA – mycophenolic acid, MHC – major histocompatibility complex, mTOR – mammalian target of rapamycin inhibitor, FKBP – FK506 binding protein, NFAT – Nuclear factor of activated T-cells, MAPK – mitogen-activated protein kinase, tgfb1 – transforming growth factor-beta 1, Cdk 2 – cyclin-dependent kinase 2, → – path continuation, – | – blocking the pathway. (note, this figure is original and not copied from another source)

4.2.1. Mechanism of action

Cyclosporine is a CNI that forms a complex with cyclophilin that inhibits the phosphatase activity of calcineurin. As a result, nuclear factor of activated T-cells (NFAT) transcription factors are not activated, and thus, the transcription of IL-2 genes in activated T cells is blocked [85]. Tacrolimus (also known as FK506) is another CNI that forms a complex with FK-binding protein 12 (FKBP12), which blocks the phosphatase activity of calcineurin [86]. The side effects of tacrolimus include nephrotoxicity, neurotoxicity, diabetogenic effects, hypertension, and increased infections [56]. MMF is a prodrug that is rapidly hydrolyzed by esterase into a pharmacologically active immunosuppressive form, MPA, after oral administration [87]. MPA controls cell proliferation by inhibiting inosine monophosphate dehydrogenase, which is involved in the de novo synthesis of guanosine nucleotides in lymphoid cells [87]. Sirolimus binds to the protein FKBP12 and forms an immunosuppressive complex that blocks the activation of the mTOR. This arrests mitosis of the cell between the G1 and S phases. Consequently, T- and B-cells activation is arrested, resulting in reduced proliferation [88]. The effect of sirolimus is synergistic with the CNIs cyclosporine [89] and tacrolimus [90].

4.2.2. Adverse effects

Cyclosporine use has been associated with nephrotoxicity [91] and hepatotoxicity [92]. Tacrolimus use has also been associated with acute nephrotoxicity requiring dose adjustment or discontinuation of the drug in 19% – 22% of patients treated with cyclosporine or tacrolimus [93]. Post-transplant diabetes mellitus incidences between 10% – 74 following treatment with tacrolimus, and to a lesser extent, cyclosporine, in combination with corticosteroids, raises the risk of morbidity and mortality [94,95]. MPA is associated with hematological side effects. T- and B-lymphocytes are most dependent on inosine monophosphate compared to other cell lines, and, as such, the proliferation of these cells is reduced by MPA [96]. MPA is also associated with gastrointestinal sequelae [97,98]. In children, gastrointestinal side effects can be severe enough to lead to a change in immunosuppressant treatment [98]. To ameliorate the side effects, an enteric-coated formulation of mycophenolate sodium has been developed and used off-label in children [98]. Sirolimus is associated with proteinuria and hematological and renal function side effects [99,100]. Cyclosporine and mTOR inhibitors like sirolimus have also been associated with an increased prevalence of dyslipidemia after pediatric renal transplant [101,102]. It should also be noted that there is a lack of consensus with the selection of induction therapies [103], such as T-lymphocyte depleting and anti-IL2 receptor monoclonal antibody treatments, which may influence the observed PD response with maintenance immunosuppressants.

4.2.3. Pharmacodynamic markers

Unlike PK, where drug concentrations are directly measured, PD monitoring is based on drug effects. Many of these markers have been measures of immune cells or their functions, such as levels of T or B lymphocytes, measurement of immune cell activation, cytokine levels, or cytokine production by immune cells. Activation of immune components could be antigen-specific or nonspecific. Alternatively, specific cellular targets of immunosuppressive medications could be monitored. This could include proteins or enzymes directly or indirectly affected by specific immunosuppressive agents.

Studies analyzing PD in pediatric renal transplantation are quite limited. However, several studies have included pediatric subjects. For example, pre-transplant assessment of immune risk using Interferon-gamma ELISPOT included pediatric patients but did not specifically report age-related results [104]. Billing et al. [105] prospectively studied 45 stable pediatric renal transplant recipients treated with cyclosporine. They measured the expression of nuclear factor of activated T cells (NFAT)-regulated genes in peripheral blood lymphocytes; NFAT-regulated gene expression was related to cyclosporine concentrations two hours after cyclosporine administration (C2) in a curvilinear fashion. There was a statistically significant correlation between NFAT-regulated gene expression with bacterial and/or viral infections requiring treatment, but a significant correlation could not be identified with cyclosporine C2 levels. This suggests that PD monitoring with NFAT genes to predict some infectious complications is superior to cyclosporine blood levels in this study population [105].

Cylex^® ImmuKnow^™ cell function assay (Cylex assay) has also been investigated for pediatric PD outcomes. This assay measures adenosine triphosphate concentrations in CD4⁺ T-cell lymphocytes and has been approved by the United States Food and Drug Administration (US FDA) as a biomarker for infection and graft survival in adults [106]. In 2005, Hooper et al. suggested modified pediatric immune response zones for patients under 12 years of age [107]. Vyas and Roberti retrospectively assessed the level of T-cell activation by Cylex assay in 44 pediatric renal transplant patients induced with Thymoglobulin (anti-thymocyte Globulin) or basiliximab and subsequent triple therapy (tacrolimus, MPA, and prednisone). The authors suggest Cylex has limited clinical utility in pediatrics, finding a statistically significant correlation between low Cylex levels with infection but no association with rejection or tacrolimus dose [108]. The clinical significance of Cylex assay results for pediatric renal transplants is currently unclear and suggests the need for additional studies in children.

Pre-transplant inosine monophosphate dehydrogenase (IMPDH) activity was measured in 28 pediatric renal transplant recipients. MPA PK and IMPDH PD showed a direct and positive relationship between MPA exposure and enzyme inhibition. The authors suggested that pre-transplant IMPDH activity may help to guide appropriate MMF dosing [109]. Importantly, IMPDH activity was lower than previously reported in adults, supporting the need for PK/PD studies in children and adolescents rather than relying on data derived from studies in adults.

4.2.4. Discrepancies between PK and PD markers

While immunosuppressants are narrow therapeutic index drugs, attaining a therapeutic window does not always correlate with the prevention of toxicities and allograft rejection. For example, Shaw et al. [110] found that MPA area under the time-concentration curve (AUC) had greater predictive power than MPA trough values for adverse outcomes. Another example is that some patients with MPA concentrations below the therapeutic window did not undergo rejection [111]. A portion of this inter-individual variability is likely due to pharmacogenetic considerations [111].

4.3. Pharmacogenetic considerations

The pharmacogenetics of immunosuppressive drugs has been studied quite extensively in adult patients [57,112–114]. While genetic factors play a role in the PK/PD of pediatric patients, some epigenetic and developmental factors may play significant roles in the inter-individual and intra-individual variability in PK, efficacy, or toxicity of the drug in pediatric patients.

4.3.1. Cyclosporine

Polymorphisms in ABCB1 and xenobiotics receptor (SXR) genes can affect cyclosporine disposition following pediatric renal transplant [115]. The age-dependent gene interaction has been demonstrated for single nucleotide polymorphisms (SNP) of ABCB1, such as C3435T and g2677, in renal transplant patients observed from 12 years of age to adulthood supporting the ABCB1 role in the developmental pharmacology of cyclosporine [115]. Another study showed that cyclosporine bioavailability in children older than eight years receiving a renal transplant increased with specific mutations in the ABCB1 gene [116]; however, the authors of this study suggested that more studies need to be done to precisely predict the effects of the gene polymorphism on cyclosporine dosing in pediatric patients.

Pediatric kidney transplant patients with a polymorphism in SXR may require cyclosporine dosage adjustment. For example, if they have a deletion/deletion haplotype of the SXR gene (g.−205–200 delGAGAAG), a lower dosage may be required to reach an effective AUC [117]. Furthermore, the age of the patient and expression of SXR mRNA in peripheral blood mononuclear cells (PBMCs) of pediatric renal transplant patients was shown to correlate directly with CYP3A4 and CYP3A5 mRNA expression in PBMCs [45]. Notably, SXR can be activated by corticosteroids which are often included in post-transplantation drug therapy [117,118].

4.3.2. Tacrolimus

Like cyclosporine, tacrolimus metabolism and bioavailability are highly dependent upon CYP3A4/5 and ABCB1 [111,119]. About one-half of the dose variability has been ascribed to the genetic variants of CYP3A5 and ABCB1 [120,121]. Almeida-Paulo and colleagues examined the role of genetic factors on the PK of orally administered tacrolimus in or for pediatric kidney transplant patients [122]. Non-expressers of CYP3A5 (CYP3A5*3/*3) had significantly higher minimal concentrations (C_min), maximal concentrations (C_max), and area under the time-concentration curve (AUC_0–24) compared to expressers (CYP3A5*1/*1 or CYP3A5*1/*3) at the same dose. When non-expressers were further investigated, it was found that elevated values of C_min, C_max, and AUC_0–24 were also associated with several SNPs in Cytochrome P450 oxidoreductases (POR) [122]. Zhao et al. analyzed the influence of CYP3A4, CYP3A5, and ABCB1 polymorphisms on tacrolimus PK in de novo pediatric renal transplant patients [123]. The authors concluded that the optimization of tacrolimus exposure in these patients should be based on weight, hematocrit levels, and polymorphism of CYP3A5. Shilbayeh also studied pediatric renal transplant patients receiving tacrolimus and found individuals who were CYP3A5 non-expressers or heterozygous for ABCB1 C3435T had adverse outcomes [124]. The effects of ABCB1 C3435T polymorphisms are not yet understood; however, additional study is underway [124].

4.3.3. Mycophenolate mofetil

Prausa and colleagues reported an association between treatment with mycophenolate and leukopenia and diarrhea in 16 pediatric renal transplant patients. Mycophenolate treatment was discontinued, or the dose was significantly lowered for these patients [125]. The authors also found that a polymorphism in UGT may increase mycophenolate drug exposure. UGT1A9-331 C and perhaps UGT2B7-900 G alleles were associated with more frequent AEs, especially leukopenia. Patients genotyped as homozygotes for UGT1A9-331 T > C developed leukopenia after mycophenolate treatment. Additionally, it has been reported that pediatric renal transplant patients with a combined polymorphism in MRP2-24 T > C and UGT1A9-440 C > T or UGT2B7-900A>G or patients with only UGT1A9-440 C > T or UGT2B7-900A>G demonstrated 2.2 and 1.7 times higher MPA AUC than patients who possessed none or only one UGT SNP [126]. Woillard and colleagues investigated the correlation between SNPs in metabolizing enzymes or transport proteins and AEs or biopsy-confirmed acute rejection in adult kidney transplant patients receiving mycophenolate sodium [127]. No association between investigated SNPs and diarrhea or biopsy-confirmed acute rejection was found. Reduced risk of anemia was observed in patients with CYP2C8 rs11572076 wild-type, while patients with UGT2B7 c.−840 G > A had a significant risk of acquiring anemia [127]. In a similar study, Jacobson and colleagues investigated the association between over 2700 SNPs and AEs in 978 kidney transplant patients receiving MPA or mycophenolate sodium [128]. None of the SNPs were associated with leukopenia, representing a false discovery rate of 20%. On the other hand, transplant patients possessing a SNP in CYP2C8 or one or two A alleles in IL12A SNP (rs568408) had a significantly increased likelihood of anemia compared to non-carriers.

4.3.4. Sirolimus

Anglicheau et al. studied 149 adult renal transplant recipients three months after initiation of sirolimus treatment. They found that non-expressers of CYP3A4 (CYP3A4*1b) and CYP3A5 (CYP3A5*3/*3) required a lower sirolimus dose than expressers [129]. Similarly, Le Meur et al. observed 47 renal transplant patients (mean age > 40 years) and found lower AUC/dose, C_max, and C_min were observed at three months carrying at least one CYP3A5*1 allele [130]. However, Emoto and colleagues’ in vitro metabolic studies suggest that it is likely that CYP3A4 contributions are much more significant than CYP3A5 [131].

4.3.5. Race and ethnic considerations

The ethnicity of the patient may be a critical factor in the PK of immunosuppressants. Differences in the PK of immunosuppressants or clinical outcomes have been observed between Caucasians and African Americans [132,133]. Poor health outcomes have been reported for African American kidney recipients treated with tacrolimus when adjustments were not made for CYP status. The bioavailability of CNIs and mTOR inhibitors appears to be significantly lower in African-Americans and to require dosage adjustment [132]. This may be partially due to the high frequency of the CYP3A5*1/*1 genotype in African Americans [134,135]. Expressers of CYP3A5 (CYP3A5*1/*1 or CYP3A5*1/*3) have lower tacrolimus trough levels, requiring increased dosage adjustments [135]. However, Pallet et al. demonstrated that unlike differences in clinical outcomes between Caucasians and African-Americans in the United States, there were no differences in 5-year post-transplant patient survival or transplanted kidney function between African Europeans and Caucasians in France [136]. Thervet and colleagues have reported that about 5% of French kidney transplant patients had CYP3A5*1/*1 genotype and potentially required tacrolimus dosage adjustment [137]. These results suggest a potential relationship between poor health outcomes reported for African American kidney recipients treated with tacrolimus when drug dosage was not adjusted and limited access to medical care. In Chinese renal transplant patients, polymorphisms in CYP3A4, CYP3A5, and ABCB1 significantly impacted the PK of CNIs [138]. Asian transplant patients also appear to have higher MPA exposure than Caucasian or African-American patients receiving an equivalent dose of mycophenolate and, therefore, required 20–46% lower MPA dosage [139].

4.4. Drug-drug interactions

Typically, a multidrug approach, using immunosuppressant agents with different mechanisms of action, allows lower dosages of single agents and reduces the severity of adverse drug effects [6,7]. In addition to immunosuppressant medication, transplant patients simultaneously take other drugs; indeed, on average, transplant patients simultaneously take ten different medications, including 2 to 3 immunosuppressants [140]. Frequent concomitant therapies associated with maintenance immunosuppression therapy include antihypertensive drugs, antiviral drugs, prophylactic antibiotic treatment, antidiabetic drugs, and anticonvulsants [3,141]. In most cases, the use of these drugs decreases over time after a kidney transplant. For antihypertensive therapy, the rates fell from 83% to 71% in recipients of a kidney from deceased donors and 78% to 63% in recipients of a kidney from live donors at two years follow up [3]. These percentages stay roughly the same at their five years follow-up. The use of prophylactic antibiotics decreased from 81% at transplant to roughly 48% at 18 months follow-up [3]. There is no apparent change out to their five years follow-up. Anticonvulsants are administered to approximately 5% of patients at transplant, which remains steady through the 5-year follow-up period [3].

Medications inhibiting or inducing CYPP450 enzymes metabolizing immunosuppressants, influencing UGT metabolism, or interacting with ABCB1 activity may significantly alter blood concentrations of immunosuppressive drugs prescribed to renal transplant patients. These drug-drug interactions may lead to the reduction or enhancement of the efficacy or toxicity of immunosuppressants. These interactions are summarized in Table 2. In pediatric patients, the magnitude of these interactions may be dependent on ontological and growth considerations.

Table 2A.

Examples of pediatric drug interactions decreasing exposure of tacrolimus, cyclosporine, MPA, Sirolimus, and Everolimus[137-140]

	Tacrolimus	Cyclosporine	MMF/MPA	Sirolimus	Everolimus
Proton Pump Inhibitors			X (MMF only)
Corticosteroids			X
Rifampin	X	X	X	X	X
Isoniazid	X	X
Anticonvulsants	X	X	X	X	X
St. John’s Wort	X	X
Cholestyramine			X
Non-nucleoside Reverse Transcriptase Inhibitors (e.g., Efavirenz)	X	X
Cyclosporine			X
Antacids with Mg(OH)2/Al(OH)3			X
Calcium Free Phosphate Binders			X

Strong to moderate drug-drug interactions are reported between maintenance immunosuppressants and macrolide antibiotics, azole antifungals, glucocorticoids, and proton pump inhibitors [142–145]. These combinations may often be prescribed to pediatric renal transplant patients. In most of these cases, co-administration of immunosuppressants with these classes of drugs should be avoided, or dosages should be significantly adjusted, requiring monitoring of drug plasma concentrations. Particular attention should be taken when herbal preparations and newer immunosuppressants are used with immunosuppressants, as interactions may not be well characterized.

4.4.1. Interactions between immunosuppressant-drug combinations

In the modern era, prophylaxis to prevent organ rejection is provided by immunosuppressants. Transplant patients will receive combinations of immunosuppressants and other drugs, and therefore, drug-drug interactions need to be considered. Unlike tacrolimus [146], cyclosporine reduces the exposure of MPA [147]. Thus, recent studies suggest a higher initial MMF dosing is needed when cyclosporine is given in combination to ensure optimum immunosuppression [148,149]. Cyclosporine also may increase the exposure of sirolimus [150]. However, in 40 stable renal transplant recipients, the addition of sirolimus to maintenance treatment of cyclosporine and prednisone was not associated with PK or nephrotoxic effects [151]. The reduction in MPA exposure occurs via inhibition of the enterohepatic recycling pathway [147], as higher doses of MMF must be given to achieve appropriate concentrations. Cyclosporine increases the exposure of sirolimus by inhibiting CYP3A4 and ABCB1 [152]. Delay of sirolimus administration until 4 hours after cyclosporine administration may reduce this increase in sirolimus exposure [152]. Sirolimus also reduces the exposure of tacrolimus [153,154], which is thought to be mediated by reducing oral absorption [154]. There may also be PD consequences for newer immunosuppressants, such as T-lymphocyte depleting and anti-IL2 receptor monoclonal antibody treatments, used in pediatric renal transplant patients [103].

4.4.2. Anti-infective

Administration of certain antibiotics, antifungal, and antiretroviral drugs may result in a significant increase in plasma concentrations of CNIs or mTOR inhibitors [155]. For example, macrolide antibiotics can inhibit CYP3A4 [155]; erythromycin and clarithromycin are known to increase calcineurin and mTOR inhibitors plasma concentration. Trofe-Clark and colleagues recommend that combinations of erythromycin and calcineurin, and mTOR inhibitors should be avoided if possible [155].

Ketoconazole and other azole-based antifungals (e.g. fluconazole, itraconazole, voriconazole) are also potent inhibitors of CYP3A4 enzyme activity. Co-administration of azole-based antifungals may increase plasma concentrations of CNIs in a dose and drug-dependent manner [155]. Simultaneous ketoconazole administration with cyclosporine leads to a rapid increase in cyclosporine plasma levels and renal toxicity or hepatotoxicity [156]. Treatment with fluconazole and CNIs may result in a delayed rise in immunosuppressant absorption [156].

4.4.3. Other medications

Co-administration of proton pump inhibitors may alter PK parameters for MPA through its pro-drug formulation, MMF. Urbanowicz et al. [157] and Sunderland et al. [158] found co-administration of proton pump inhibitors delayed onset of MPA exposure after parenteral administration of MMF, likely due to the reduction of the transformation of the prodrug MMF to MPA that occurs at low pH. Sunderland et al. [158] also found that co-administration enteral pantoprazole with enteric-coated mycophenolate sodium, which is transformed at neutral pH, decreased the Tmax of MPA. Glucocorticoids may also decrease renal transplant patient exposure to MPA by induction of liver UDP-glucuronosyltransferase [142,159,160]. Moderate interaction of MPA and the CYP3A4 inducer, rifampin, has been reported to require increased doses of MMF [156]. It has been suggested that rifampin, isoniazid, phenobarbital, phenytoin, and verapamil co-administration with CNIs or mTOR inhibitors may alter drug exposure in renal transplant patients [142]. Table 2 highlight some of the drug-induced changes in immunosuppressant exposure. As a potent inducer of CYP3A4, rifampin stimulates the rapid clearance of CNIs and mTOR inhibitors [156]. Moderate interaction of MPA and rifampin has been reported requiring increased doses of MMF [156]. As a result, the combined treatment with rifampin and calcineurin or mTOR inhibitors should be avoided or the concentrations monitored. Calcium channel blockers, such as verapamil, nicardipine, nifedipine, may also increase tacrolimus concentrations and toxicity through partial inhibition of CYP3A4 [142,161,162].

5. Bioequivalence

There are many generic versions of immunosuppressants [140] (Table 3). Generic substitution for brand name immunosuppressants has been shown to result in variable concentrations that may negatively impact a patient’s therapy[163]. Drugs that meet generic approval have an intra-patient variability of ≤ 30% in C_max and AUC [164]. For example, Qazi et al. compared Neoral^® to generic cyclosporine and demonstrated that nearly 20% of patients on the generic had to have dose adjustments to achieve therapeutic trough levels after switching from brand to generic [165]. Unique formulation concerns also arise in pediatric patients. Not every immunosuppressant is available in a liquid formulation. For patients unable to swallow, this necessitates the creation of an extemporaneous formulation by a pharmacy, for which the PK effects, including bioavailability, are usually unknown. Typical bioequivalence (BE) studies are done in 24–36-year-old healthy, male volunteers [140]. In contrast, transplant patients represent a distinct and heterogeneous population. Commonly, transplant recipients exhibit poor drug absorption due to comorbidities [140] that cause a significant degree of PK variability.

Table 2B.

Examples of pediatric drug interactions increasing exposure of tacrolimus, cyclosporine, MPA, Sirolimus, and Everolimus [137-140]

	Tacrolimus	Cyclosporine	MMF/MPA	Sirolimus	Everolimus
Calcium Channel Blockers	X	X
Azole-based Antifungals	X	X
Macrolide Antibiotics	X	X		X	X
Cyclosporine				X	X
Protease Inhibitors (e.g., Ritonavir)	X	X
Proton Pump Inhibitors				X	X
Grapefruit Juice	X	X		X	X

6. Dose individualization and therapeutic drug monitoring

Individualization of patients’ immunosuppressant therapy is often required to optimize the balance between therapeutic efficacy and drug-induced toxicity. Therapeutic drug monitoring (TDM) has been the most effective tool in adjusting for inter-individual variability in immunosuppressant efficacy or toxicity in renal transplant patients. A plethora of factors contributes to the inter-individual variability [146,166–171], which is further perplexed in pediatric patients due to additional factors such as developmental changes affecting drug PK (adsorption, distribution, metabolism, and excretion), and inadequacy or heterogeneity of the available data to establish individualized therapy in pediatrics [172].

Immunosuppression in children receiving a renal transplant is separated into two phases: induction to prevent acute rejection and maintenance to maintain long-term immunosuppression. Patients are generally treated with high-dose immunosuppressants during the first few months after transplantation when the risk of acute rejection is the highest. Individualized MMF dosing based on body surface area may be used as a maintenance immunosuppressant in pediatric renal transplant patients [3,173]. As the dose-normalized AUC of MPA varies by 10-fold in pediatric and adult renal transplant patients, personalized dosing is crucial for this drug [174]. A delicate balance between under- and over-immunosuppression in MMF-treated patients will be challenging to achieve. Proper estimation of MMF dose-interval and AUC through a limited sampling strategy might be useful to avoid acute rejection. However, an appropriate TDM protocol for MMF maintenance therapy is difficult to formulate and is not routinely utilized in renal transplant patients. Similarly, another variability source is the unbound fraction of MPA in plasma that changes based on renal function, concomitant steroid use, hypoalbuminemia, acidosis, and hyperbilirubinemia [174,175].

Therapeutic drug monitoring of cyclosporine and tacrolimus is also crucial in pediatric renal transplant recipients. While the AUC of cyclosporine is a better predictor of total body exposure, trough cyclosporine concentration monitoring is widespread because of the convenience and acceptable correlation with drug exposure [176]. Patient age, time after drug administration, liver function, hematocrit, albumin blood concentrations, and concomitant steroid administration are the most critical factors contributing to the inter-individual variability in tacrolimus PK. Tacrolimus exposure is correlated to the risk of acute rejection and a decline in graft function due to renal CNI toxicity [177]. However, the AUC of tacrolimus is the best predictor of drug exposure; a limited sampling strategy with 3–4 samples in the first 4 hours after drug administration has been useful in optimizing the dosing of tacrolimus [178,179]. In clinical practice, however, trough levels are most commonly used to monitor tacrolimus exposure, unless there is a suspicion that drug exposure may be unusual or if a patient is also on interacting agents (e.g. anticonvulsants, antifungals, combinations). TDM of sirolimus and its derivative, everolimus, is also suggested because both drugs show significant PK variabilities and age dependency in clearance [180–183].

7. Conclusions

As outlined in this review, immunosuppressant therapies remain the mainstay treatment for solid organ and bone marrow transplants. These drugs are widely used in children. The immunosuppression regimen choice depends on the institution, transplant type, and individual donor and recipient risk factors. Typically, a multidrug approach, using immunosuppressant agents with multiple mechanisms of action, allows lower doses of single agents and reduces the severity of adverse drug effects. Immunosuppressant agents used in multidrug therapies include cyclosporine, tacrolimus, sirolimus, and MMF. Cyclosporine and tacrolimus inhibit calcineurin, sirolimus acts as an mTOR inhibitor, and MMF, a prodrug of MPA, inhibits IMPDH, the rate-limiting step for de novo guanosine nucleotide synthesis. These variable mechanisms of action all decrease proliferation of B- and T-lymphocyte cells to prevent graft rejection and diminish transplant complications. As outlined in this review, many factors affect immunosuppressant drug concentration, including drug-drug interactions, alteration of efflux pump transporters and enzyme activity by concomitant medications, genetic polymorphisms, age, drug-food interactions, and disease. These drugs have a narrow therapeutic index and therefore require TDM to ensure safe and successful therapy. There are many generic versions of various immunosuppressant agents. However, the criteria for demonstrating that a generic immunosuppressant is therapeutically equivalent to a brand name comparator are still not well defined and have been the subject of multiple studies.

8. Expert opinion

Each year in the United States, nearly 29,000 children and adults who receive a solid organ or bone marrow transplant are given immunosuppressant therapies as their mainstay treatment [184–186]. Among these patients, 17,000 received kidney transplants in 2014 alone [187]. Special consideration should be given to the pediatric populations as developmental changes may influence immunosuppressant effectiveness and have long-term consequences. Advances in immunosuppressive therapy and overall renal transplant patients’ clinical care allow pediatric kidney recipients to transition from childhood to adulthood successfully. The choice of an immunosuppression regimen is dependent on institution, transplant type, and individual donor and recipient risk factors. These drugs are associated with a narrow therapeutic index and require TDM to ensure safe and successful therapy [188–190]. Many factors affect immunosuppressant drug concentration, including drug-drug interactions, alteration of efflux pump transporters, metabolizing enzyme activity, genetic polymorphisms, age, drug-food interactions, existing conditions, and acquired disease [191]. These factors need to be accounted for to provide additional stratification of dosing recommendations in pediatric kidney transplant patients.

Bioequivalence (BE) is an area of continued investigation; not all generic immunosuppressants have similar bioequivalences to their brand comparators. Children are rarely considered when undertaking generic drug substitution evaluations or drug studies in general, despite evidence that pediatric patients require significantly higher weight-based doses of tacrolimus to maintain drug exposure that is similar to adults [78]. For these reasons, both the National Kidney Foundation and the American Society of Transplantation support BE studies of immunosuppressants in the target population [192,193]. As outlined by Tsipotis et al. [194], not all generic immunosuppressants have similar bioequivalences to their brand comparators. There needs to be a push toward regulatory oversight of studies investigating narrow therapeutic index immunosuppressants to compare branded versus generic and new formulations (e.g. enteric coating mycophenolate) in pediatric patients.

In many circumstances, results from efficacy and safety studies between generic and brand drugs are inconclusive [194]. Questions related to the generic versions of immunosuppressant agents used in pediatric patients remain for these drugs having a narrow therapeutic index which may lead to severe adverse drug reactions or therapeutic failure. In post-transplant, the prospect of graft rejection is a critical concern because this represents therapeutic failure and is potentially life-threatening. In general, narrow therapeutic index drugs have been shown to have low within-subject variability. Interestingly, of the four immunosuppressant drugs outlined above with potential concerns associated with their narrow therapeutic index, only tacrolimus currently carries a specific treatment recommendation as a narrow therapeutic index drug based on BE studies. There have been multiple guidelines released, but even with new recommendations such as those from the US FDA and in the drug labels, it is unclear whether the narrowed limits for BE improve the likelihood of a generic drug performing similarly to the innovator product especially in pediatric patients. BE studies in the transplant population are complicated and require a much larger sample sizes to reach significant statistical power than in a healthy group [195]. Population PK modeling can be used to overcome the small sample size of transplant patients by evaluating PK profiles and PD markers to assess BE. In younger populations, these studies should include pharmacogenetics considerations as well as growth and development which is highly variable in children and can modify the relation between genotype and phenotype.

The criteria for demonstrating that a generic immunosuppressant is therapeutically equivalent to a brand name comparator are under investigation. Concerning these agents, there are general BE considerations such as those outlined in the 2003 US FDA guidelines for demonstrating BE. A generic with the same dosage form, active pharmaceutical ingredients, and dose strength can be considered bioequivalent to a brand name drug if the AUC and C_max fall within 80– 125% of the reference values along with the 90% confidence intervals of each value [196]. These guidelines suggested the same cutoffs for drugs having a narrow therapeutic index where small differences in blood concentrations or doses of the drug may lead to severe adverse drug reactions or therapeutic failure [196]. Based on concerns as to whether the standard guidelines were appropriate [197], in 2011, the US FDA recommended a new set of BE limits for generic drugs with a narrow therapeutic index based on the within-subject variability of the reference drug [198]. Specifically, if the reference variability is ≤10%, then the reference-scaled BE must be within a limit of 90–111%. If the reference variability is >10%, then the BE limits are reference-scaled with limits set at 80–125% [198]. Narrow therapeutic index drugs generally have low within-subject variability and will often fall into the first category [198]. The underlying assumption of the BE criteria is that formulations with similar PK profiles will have similar efficacies [163]. Encouragingly, one recent prospective study found generic tacrolimus to be bioequivalent to generic formulation in adult kidney and liver patients [199]. While this may be suitable for most drug classes, this variability may be too large for narrow therapeutic index immunosuppressant therapies, particularly in pediatric populations, which may require increased dosing to reach therapeutic concentrations. Therefore, even with these new recommendations, it is unclear whether the narrowed limits improve a generic drug’s likelihood of achieving similar efficacy to the innovator product, particularly in the pediatric population [200]. This is the US FDA definition that a generic with the same dosage form, active pharmaceutical ingredients, and dose strength can be considered bioequivalent to a brand name drug if the AUC_0–24 and C_max, along with the 90% CIs of each value, fall within 80–125% of the reference values. Despite the US FDA guidelines and other literature, there remain concerns that there may not always be BE between a generic and a brand name drug.

Pediatric patients are rarely considered when undertaking generic drug substitution evaluations. Clinical studies in pediatric patients have demonstrated that children require 2–4 times the tacrolimus dose to maintain similar drug exposure as adults. For these reasons, the National Kidney Foundation and the American Society of Transplantation support BE studies of immunosuppressants in the target population. However, BE studies in the pediatric transplant populations are challenging and require a much larger sample size to reach significant statistical power compared to a healthy group of adults. Population pharmacometric modeling can overcome the small sample size of transplant patients by evaluating PK profiles, PD markers, and significant covariates; this helps move the dosing of these agents toward a precision medicine approach.

Population pharmacometric modeling uses applied mathematics and statistics to understand the PK and PD of a drug. The introduction of population pharmacometric modeling and simulation has greatly improved the models’ ability to estimate drug PK and PD parameters across drug classes and account for the effects of many variables between patients within many disease states. Traditional PK studies could not provide parameters for sub-populations such as children and those with a disease-state affecting drug PK, such as organ transplants. By utilizing data from TDM to predict typical values and variability of PK parameters, these models allow for superior optimization of drug dosing and more in-depth knowledge of drug PK in transplant patients. Population methods are becoming more widely accepted and are applied to PK, PD, and models linking biomarkers to clinical outcomes. Utilizing PK/PD modeling in developing clinical dosing guidelines allows the full breadth and depth of the individual PK to be determined while preserving the individual variability. Over recent years, studies for mainstay immunosuppressive drugs have been published in several papers. These have included PK models developed to propose optimal dosing of MMF and cyclosporine to prevent organ rejection. The current models can be further refined and focused by adding patient data from TDM combined with the patients’ clinical characteristics.

Understanding the multiple factors that influence immunosuppressive agents used to treat pediatric patients’ post-transplantation is a priority. There are many unique factors, such as specific pediatric PK, PD, and developmental changes, where considerations differ from those in adults. The need to provide effective and efficient therapy is critical to extend host and graft survival. The use of these drugs for immunosuppression in pediatric transplant patients requires continued discussion and consideration of the factors which influence successful treatment. Clinicians need to implement the best treatment plan utilizing a precision medicine approach for their patients.

Table 3.

Summary of available maintenance immunosuppressants, routes of administration and formulations approved by the FDA according to information available at https://www.accessdata.fda.gov/drugsatfda

Drug Class	Immunosuppressant	Administration Route	Formulation (Approved Brand/Generic)
CNI	Cyclosporine	Intravenous	West-Wared Pharms Int, Luitpold, *Sandimmune® (Novartis)
		Oral (capsule)	*Neoral® (Novartis), Sandoz, *Gengraf® (Abbvie), Apotex, Mayne Pharma, IVAX Sub Teva Pharms, *Sandimmune® (Novartis)
		Oral (solution)	*Neoral® (Novartis), Abbvie, Apotex, Mayne Pharma, IVAX Sub Teva Pharms, Wockhardt, *Sandimmune® (Novartis)
	Tacrolimus	Intravenous	*Prograf® (Astellas)
		Oral (capsule)	*Prograf® (Astellas), Dr Reddys Labs Ltd, Mylan, Strides Pharma, Panacea Biotec Ltd, Accord Healthcare
		Oral (capsule-extended release)	*Astagraf XL® (Astellas)
Inosine-monophosphate dehydrogenase inhibitor	Mycophenolate Mofetil	Intravenous	*Cellcept® (Roche Palo)
		Oral (capsule)	*Cellcept® (Roche Palo) Sandoz, West-Ward Pharms Int, Accord Healthcare, Teva Pharms, Mylan, Strides Pharma, Vintage Pharms LLC, Accord Healthcare, Apotex Corp, Jubilant Cadista, Alkem Labs Ltd
		Oral (tablet)	*Cellcept® (Roche Palo), Sandoz, West-Ward Pharms Int, Accord Healthcare, Teva Pharms, Mylan, Strides Pharma, Vintage Pharms LLC, Apotex, Jubilant Cadista, Alkem Labs Ltd
			*Cellcept® (Roche Palo), Alkem Labs Ltd
		Oral (suspension)	Par Sterile Products, Mylan Labs Ltd, Akorn Inc
	Mycophenolate Mofetil HCl	Intravenous
mTOR Inhibitors	Sirolimus	Intravenous
		Oral (tablet)	*Rapamune® (PF Prism CV), Dr Reddys Labs Ltd, Zydus Pharms USA Inc
		Oral (suspension)	*Rapamune® (PF Prism CV)
	Everolimus	Oral (tablet)	*Afinitor® (Novartis), *Zortress® (Novartis)
		Oral (tablet, for suspension)	*Afinitor Disperz® (Novartis Pharm)

CNI – calcineurin inhibitors, mTOR – mammalian target of rapamycin

^*– Brand formulation

Article highlights.

• Developmental differences in pediatric renal transplant patients influences treatment plan decisions and overall patient outcomes.

• Maintenance phase immunosuppressant protocols have evolved over the last several decades.

• Toxicities and adverse drug reactions associated with immunosuppressant therapy can be minimized by controlling therapeutic target concentration attainment based on therapeutic drug monitoring.

• Individual patient factors (e.g. age, weight, hematocrit, and pharmacogenetics) should be considered when implementing immunosuppressant treatment plans.

• Studies comparing the bioequivalence of generic formulations to brand formulations are ongoing.