Objectives: This review focuses on the current limited evidence of graft function and graft survival in various immunosuppressive regimens involving mammalian target of rapamycin inhibitors with or without calcineurin inhibitors.

Materials and Methods: We evaluated the current literature for describing the role of mammalian target of rapamycin inhibitors as an alternative to calcineurin inhibitors by searching the PubMed, EMBASE, Cochrane, Crossref, and Scopus databases using medical subject heading terms.

Results: Our detailed analyses of all relevant literature showed use of mammalian target of rapamycin inhibitor-based de novo regimens, early calcineurin inhibitor withdrawal with subsequent introduction of mammalian target of rapamycin inhibitor-based regimens, and late conversion from a calcineurin inhibitor-based regimen to mammalian target of rapamycin inhibitor-based regimens. Notably, early calcineurin inhibitor withdrawal with subsequent introduction of mammalian target of rapamycin inhibitor-based regimen seemed to be a more practical and realistic approach toward immuno-suppressive treatment of renal transplant recipients. However, in view of the high rejection rate observed in these studies, it is advisable not to offer these regimens to patients with moderate to high immuno­logic risk.

Conclusions: The present evidences suggest that treatment with mammalian target of rapamycin inhibitors allows early and substantial calcineurin inhibitor minimization. The mammalian target of rapamycin inhibitors everolimus and sirolimus are preferred due to their complementary mechanisms of action and favorable nephrotoxicity profile, which have opened the way for calcineurin inhibitor reduction/withdrawal in the early posttransplant period.

Key words : Adverse events, Kidney transplantation, mTOR inhibitors

Introduction

Kidney transplant has improved survival rates in patients with chronic kidney disease. The re­structuring of transplant immunology over the past half century has continuously improved 1-year graft survival rates and acute rejection.¹ Since their introduction in 1980, the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus have been highly regarded, as evidenced by the significantly better outcomes notable with these agents, that is, more than 90% 1-year graft survival while maintaining an acute rejection rate of less than 20%.² However, the excellent results of short-term allograft survival have not translated into long-term graft survival. Long-term survival continues to show a slow, steady decline with some categories falling below 50% over 10 years.^3,4 Several studies have demonstrated that the primary cause of late graft loss is chronic allograft nephropathy and death with functioning graft with reported incidences of 40% and 43%. The most frequently reported causes of death with functioning graft are cardiovascular complications and cancer.⁵ Chronic allograft nephropathy is a commonly used term to describe a pattern of interstitial fibrosis and tubular atrophy thought to represent the terminal spectrum of end-stage renal injuries after transplant. The commonly associated risk factors for chronic allograft nephropathy are divided into immunogenic and nonimmunogenic factors. Immunogenic factors include acute rejection, retransplant, HLA antibodies, and non-HLA antibodies, whereas conventional nonimmunogenic factors are associated with is­chemic-reperfusion injury, transplanted nephron mass, nephrotoxic drugs such as CNI, hypertension or hyperlipidemia, cytomegalovirus, and hyperfil­tration.^6,7

In 2005, the BANFF classification of renal allograft biopsies was introduced, and the term “chronic allograft nephropathy” was replaced with interstitial fibrosis/tubular atrophy. The term “interstitial fibrosis/tubular atrophy” must be used cautiously and only be labeled after all attempts to identify a specific cause have been exhausted.⁸

Calcineurin inhibitor-incited nephrotoxicity was described as the most significant cause of long-term graft failure (in 96.8% of allograft biopsies)⁹. Cal­cineurin inhibitors create imbalance by increasing the yield of vasoconstrictors, such as endothelin and thromboxane, and decreasing the production of vasodilators, such as nitric oxide, prostacyclin, and prostaglandin E2. Calcineurin inhibitors directly affect juxtaglomerular cells or indirectly affect renal hemodynamics to activate the sympathetic neural system and the renin-angiotensin system. Calci­neurin inhibitors cause vascular remodeling by promoting vascular smooth muscle cell necrosis and hyaline deposition, which leads to the narrowing of the lumen of a vessel. In summary, CNIs promote platelet aggregation, resulting in a prothrombotic state that increases the peril of the thrombotic microangiopathy. They further cause renal ischemia that in the final analysis results in the fibrosis.^7,10 Calcineurin inhibitors also promote transforming growth factor-β and up-regulate apoptotic genes, which further leads to interstitial fibrosis. Furthermore, CNIs give unmediated tubular toxicity, kindling tubular atrophy.

The typical histologic patterns, including nodular arteriolar hyalinosis, tubular atrophy, luminal nar­rowing, tubular vacuolization, interstitial fibrosis, global or focal segmental sclerosis, and microcalci­fications, were used to define CNI nephrotoxicity.¹¹ In a study from 2004, Nankivell and associates reported that more than 50% of kidney allograft biopsies unveiled attestation of chronic CNI toxicity and after 10 years 100% exhibited this framework of CNI injury.⁴ This explains why the small early acute rejection rates have been accomplished using CNI therapy, yet are not accompanied by any long-term benefits. Calcineurin inhibitors have been linked with the development of multiple cardiovascular disease risk factors, including hypertension, hyperlipidemia, and new-onset diabetes mellitus after transplant.¹²

To minimize CNI-induced nephrotoxicity, various other CNI-free, CNI low-dose, or nonnephrotoxic immuno­suppressive drug-based protocols have been introduced. However, the requirement to limit CNI-induced nephrotoxicity must be weighed against the anticipated increased risk of acute or chronic antibody-mediated rejection seen with low-dose CNI. Powerful nonnephrotoxic agents, such as the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus, have emerged with an immunosuppressive mode of action complementary to that of CNIs, providing the rationale for their combined clinical use.^13,14

The calcineurin inhibitors tacrolimus and cyclosporine bind to intracellular proteins called FK506 binding protein and immunophilins; these combinations further block the effects of calcineurin inhibitors, catalyzing the intracellular processes associated with the activation of T lymphocytes. This results in reduced production of interleukin 2, inhibiting T-cell proliferation.^15,16 According to the Kidney Disease Improving Global Outcomes 2009 clinical practice guidelines, the combination of tacrolimus with an antiproliferative agent, with or without corticosteroids, is the most favored first-line CNI for initial maintenance therapy. Thus, tacrolimus-based regimens are the most commonly used regimens, with 80% of initial immuno­suppression and 70% of maintenance immunosup­pression received by renal transplant recipients.¹⁷

Mammalian target of rapamycin inhibitors such as sirolimus and everolimus also bind to FK506 binding proteins, and this complex also reduces T-cell activation by blocking growth factor-mediated cell proliferation in the cellular response to alloantigens in the cell cycle.¹⁴ The distinct mechanism of action and favorable nephrotoxicity of mTOR inhibitors have lured many clinicians to use them as a replacement to CNIs in kidney transplant. In addition, there is a minimal pharmacologic inter­action between tacrolimus and mTOR inhibitors, whereas cyclosporine and mTOR inhibitors exhibit more pronounced interactions, resulting in higher blood levels of mTOR inhibitors. However, for higher loading doses of everolimus, its concentration should be monitored in combination with low-dose tacro­limus to prevent increased risk of rejection. At present, there are limited clinical studies in the literature to comment on the use of everolimus or sirolimus in tacrolimus minimization strategies. Available evidence suggests that treatment with everolimus allows early and substantial tacrolimus minimization when used with basiliximab induction and corticosteroids. The recent studies have reported a significant increase in estimated GFR following the switch to sirolimus from CNIs 2 months after transplant.^18,19

The main aim of this review is to focus on the reported current limited evidence of graft function and graft survival in various immunosuppressive regimens involving mTOR inhibitors with or without CNI in different randomized clinical studies.

Materials and Methods

A comprehensive systematic literature review was performed following registration in PROSPERO, an international database of prospectively registered systematic reviews (registration number CRD42016048519). We searched all of the published articles in the National Library of Medicine Database (PubMed), as well as the EMBASE, Cochrane, Crossref, and Scopus databases on September 15, 2016, for original articles describing the role of mTOR inhibitors as an alternative to CNIs. The search covered the period from 2001 (the year of the first reported early cyclosporine withdrawal with sirolimus in the literature) to September 15th, 2016. The search was carried by using the following medical subject headings (MeSH) terms: calcineurin inhibitors, tacrolimus, cyclosporine, mTOR inhibitors, sirolimus, everolimus, kidney transplant, adverse events, graft rejection, and graft survival. The initial search yielded a total of 112 manuscripts. After careful evaluation, 92 articles were excluded. The remaining 20 articles were considered, and full text was obtained for each.

Inclusion criteria
Original studies available in English and published between 1990 and September 2016 were included. Only studies that systematically and quantitatively assessed the graft function and graft survival in various immunosuppressive regimens involving mTOR inhibitors with or without CNIs in different randomized clinical studies were analyzed. Publication types included comparative studies and retrospective and prospective studies. We excluded other publication types, such as editorials, reviews, and letters.

Data extraction
Using a standardized quality assessment tool and prespecified inclusion and exclusion criteria, we analyzed all papers with empirical studies. This systematic review was performed using the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines and was registered in PROSPERO, an international database of prospectively registered systematic reviews (Figure 1 and Table 1). A total of 20 articles matched our described inclusion criteria.

Results

The quality of all of the included articles were assessed by QUADAS-2 (quality assessment for studies of diagnostic accuracy) and prespecified inclusion and exclusion criteria. QUADAS-2 is an evidence-based bias assessment tool to assess the quality of diagnostic accuracy studies in a systematic review.

Twenty peer-reviewed studies were included in this analysis; 10 studies were from single centers, whereas the remaining 10 were multi-institutional studies. We reviewed each study comprehensively, and data were extracted for the outcomes, such as patient safety, exposure-response relationships, ad­verse events, and various shortcomings or weaknesses to improve the graft functioning and long-term survival.

Publication bias is always a concern in systematic reviews because all investigations that take place have not been published. Studies with significant results are more likely to be published and are more likely to be published in a higher impact journal versus studies with negative results. Moreover, well-controlled and properly performed studies are less likely to achieve significance.

Discussion

The introduction of mTOR inhibitors, such as eve­rolimus and sirolimus, is a difficult decision as it should be done after the period of highest im­munologic risk and wound infection but before the initiation of CNI-related renal toxicity (Tables 2 and 3).

Mammalian target of rapamycin-based initial regimen with complete avoidance of calcineurin inhibitors (calcineurin inhibitor-free regimen)
To date, several studies have been done to assess the transplant outcomes with a CNI-free initial regimen. Notable studies are the ORION trial by Flechner and associates (2007) and SPIESSER study (2012). The ORION trial showed favorable outcomes, as sig­nificantly better renal functioning with comparable biopsy-proven acute rejection rate (BPAR) toward mTOR inhibitors. Here, they compared an mTOR-inhibitor sirolimus with a CNI-based regimen.¹² However, in an another study by the same group in 2011, the group found a higher glomerular filtration rate (GFR) in the CNI-free group but at the expense of significantly higher BPAR.²⁰

Similarly, other randomized trials have reported significantly higher acute rejection rates than with CNI-based regimens, which could be minimized with an introduction of an induction agent such as a lymphocyte-depleting antibody. The usual first-year rejection rates, as reported through various CNI-based regimens, are less than 7%. In the SPIESSER study, the investigators compared cyclosporine, mycophenolate mofetil (MMF), and a steroid combination with sirolimus, MMF, and a steroid combination after induction with a lymphocyte-depleting agent (such as antithymocyte globulin). The reported outcomes at 1 year in terms of efficacy, graft functioning, and survival were comparable in both groups.²¹

Mammalian target of rapamycin-based initial regimen with low-level of calcineurin inhibitors (calcineurin inhibitor minimization regimen)
The tacrolimus and mTOR inhibitors have shown minimal pharmacologic interaction, whereas cy­closporine and mTOR inhibitors exhibit a more pronounced pharmacodynamic interaction, resulting in higher blood levels of mTOR inhibitors. Therefore, concentration monitoring has been needed to prevent rejection and toxicity risk. Limited studies have been done so far to assess the transplant outcomes with an mTOR inhibitor-based initial regimen with CNI minimization, with the notable ones being the ASSET study (2012) and the studies of Russ and associates (2003), Lo and associates (2004), Hamdy and associates (2005), Ekberg and associates (2007), Guerra and associates (2011), Chhabra and associates (2012), and Takahashi and associates (2013).^22-29

In 2012, Langer and associates in their ASSET study compared de novo introduction of very-low-dose tacrolimus (1.5-3 ng/mL) and everolimus (3-8 ng/mL) with low-dose tacrolimus (1.5-3 ng/mL) and everolimus (3-8 ng/mL). These have been used in a combination with oral steroids following basiliximab induction in renal transplant patients. They reported a significant improvement in the estimated GFR in the low-dose tacrolimus group, although there was no marked improvement seen regarding graft and patient survival. The incidence of BPAR episodes were significantly higher in very-low tacrolimus group (18.7% vs 7.7%; P = .01).²²

In 2013, Takahashi and associates studied the early introduction of low-dose cyclosporine and everolimus (baseline level or 0-h blood level of 3-8 ng/mL) with standard-dose cyclosporine and MMF (2 g/d) in combination with oral steroids following basiliximab induction in renal transplant patients. In the low-dose cyclosporine group, levels were gradually reduced from 100 to 200 ng/mL to 25 to 50 ng/mL over 6 months, whereas in the standard dose group, cyclosporine was reduced from baseline level of 200 to 300 ng/mL to 100 to 250 ng/mL in month 2 and further maintained at the same level. The group did not report any significant improvement in terms of estimated GFR, graft rejection, and graft and patient survival. The incidence of wound healing events was 200% higher in the everolimus group than in the MMF group (39.3% vs 11.5%).²⁹

When outcomes in available literature are considered, mTOR inhibitor-based initial protocols that are either free of CNI or have CNI minimized are not an acceptable choice due to the higher number of adverse effects and acute rejection rates.

Mammalian target of rapamycin inhibitor-based early conversion from calcineurin inhibitor regimen
To reduce the nephrotoxic effects of CNI drugs, different regimens with a lower dose or avoidance of these drugs have been tried. In the presence of heightened risk of rejection associated with de novo use of CNI-free protocols, they are presently recommended. The optimal timing regarding the switch to mTOR inhibitor-based immunosup­pression is not clear. Various studies have reported evidence toward a benefit of conversion to mTOR inhibitors within 6 months of transplant, whereas some evidence shows that a conversion after month 6 has limited benefits. This lack of benefit after late conversion could be because of the development of CNI nephrotoxicity in the allograft well before the initiation of conversion.

Mammalian target of rapamycin inhibitor-based early conversion
In this approach, mTOR inhibitors are introduced within a period of 2 weeks to 6 months of transplant, a period when increased risks for rejection and wound infection have passed. Several studies that have assessed transplant outcomes with an mTOR inhibitor-based initial conversion regimen include the CENTRAL trial (2013), the ZEUSS trial (2014), the Spare the Nephron trial (2011), the SMART trial (2010), the CONCEPT trial (2012), and the study of Bansal and associates33-38 (Tables 4 and 5).

In 2013 in the CENTRAL trial, Mjornstedt and associates investigated early conversion from cyclosporine to everolimus in week 7 posttransplant. They randomized 202 patients to receive everolimus (3-8 ng/mL) versus cyclosporine (0-h blood level of 75-200 ng/mL for 2 wk, which was then reduced and further maintained at 50-150 ng/mL) in combination with oral steroids and MMF. They reported a significant improvement in GFR in the everolimus group (51.2 ± 14.1 vs 48.8 ± 15.4 mL/min; P = .012). The reported incidence of acute rejection was significantly higher in the everolimus group than in the cyclosporine group (27.5% vs 11.0%; P = .004). Similarly, significantly more patients in the everolimus group had serious adverse effects, including anemia, hyperlipidemia, proteinuria, acne, and mouth ulceration.³³

In the 2014 multicenter randomized ZEUSS study done by Budde and associates, early conversion from cyclosporine to everolimus at 4.5 months post­trans­plant was considered, with 269 patients randomized into 2 groups to receive either everolimus with MMF or to have maintained and then gradually tapered lower doses of cyclosporine with MMF. The investigators reported a statistically significant improvement in renal functioning (estimated GFR) for the everolimus group (71.8 vs 61.9 mL/min; P = .004). The reported incidence of BPAR was higher in the everolimus group (13.9% vs 7.5%; P = .09), although both groups were quite comparable regarding graft and patient survival.³⁴

In 2011, Weir and associates performed the Spare the Nephron Trial, in which 299 patients were randomized into 2 groups approximately 115 days after renal transplant. In the first group, sirolimus with MMF was introduced, whereas the second group was maintained on CNI and MMF. The investigators reported significantly better im­provement in renal function (GFR) in the sirolimus group (75.5 ± 19.2 vs 71.2 ± 23.5 mL/min; P = .04). There was no significant change regarding patient and graft survival. However, patients in the sirolimus group reported a significantly higher number of adverse effects, including hyperlipidemia, diarrhea, and mouth ulcers.³⁵

In a multicenter randomized SMART trial by Guba and associates in 2010, early conversion from cyclosporine to sirolimus only 10 to 24 days after renal transplant was studied in 141 patients randomized into 2 groups to receive sirolimus with MMF and steroids or maintained on gradually tapered lower dose of cyclosporine with MMF and steroids. The investigators reported a statistically significant improvement in renal function (estimated GFR) and serum creatinine levels for the sirolimus group (71.8 vs 61.9 mL/min; P = .004; and 1.51 ± 0.59 vs 1.87 ± 0.98 mg/dL; P = .004). The incidence of BPAR was similar in both groups (17.4% vs. 15.5%; P = not significant). Similarly, graft and patient survival rates were quite comparable. Patients in the sirolimus group reported a significantly higher number of adverse effects such as hyperlipidemia and acne, although cytomegalovirus viremia was a signifi­cantly lower number.³⁶

In the 2009 CONCEPT study by Lebranchu and associates, sirolimus was introduced in place of cyclosporine 3 months after transplant. After 1 year, the sirolimus group recorded significant improve­ments in estimated GFR (68.9 vs 64.4 mL/min). Biopsy-proven acute rejection rate was noted in 16.8% of patients in the sirolimus group and in 8.2% in the cyclosporine group. Adverse effects, including diarrhea, new-onset diabetes mellitus, acne, aphthous stomatitis, and high triglyceride levels, were significantly higher in the sirolimus group.³⁷

In 2013, Bansal and associates investigated the effects of early switching from CNI-based regimens to sirolimus. Similar to other trials, they also studied the effects of these regimens on renal function, graft rejection, patient survival, and adverse effects. The authors also investigated the role of these drugs in the CD4+CD25+ T-regulatory cell population. These cells play an immunomodulatory role by down-regulating the alloantigen-specific immune response. Regulatory T-cell production and continuation necessitate T-cell activation by binding its receptor and interleukin 2 signaling. However, the effects of immunosuppressive medications on regulatory T-cell homeostasis are different as cyclosporine inhibits the conversion of CD4+CD25 naive T cells to CD4+Foxp3+ regulatory T cells.38 In contrast, Sengundo and associates in 2006 reported an increase in the number of these cells in response to sirolimus.³⁹

Sixty transplant recipients were randomized 3 months after surgery to receive sirolimus (initial level of 8-15 ng/mL) or a CNI-based regimen. The sirolimus group had significant improvements in renal function in terms of GFR (88.9 ± 11.8 vs 80.6 ± 16.5 mL/min; P = .04) and serum creatinine levels (1.14 ± 0.17 vs 0.99 ± 0.11 mg/dL; P = .04). The reported incidence of acute rejection was lower in the sirolimus group than in the cyclosporine group (0% vs. 6.8%).

The reported adverse effects (proteinuria, hyper­lipidemia, mouth ulcers, and new-onset diabetes mellitus) were similar in both groups. Interestingly, patients in the sirolimus group reported a significantly higher incidence of enthesitis (17.39% vs. 0%; P = .000). They also reported a statistically significant increase in the regulatory T-cell population compared with control (39.5 ± 16.70 vs 28.1 ± 18.20 mL/min; P = .04). A study by Korczak-Kowalska and associates in 2007 heralded no increment in the regulatory T-cell population in contrast to healthy individuals in reaction to sirolimus but was significantly appreciative compared with cyclosporine-treated patients. This result seems to be unaffected by transplant duration.40 In a recent study (2013), Carroll and associates reported a similar increase in the regulatory T-cell population in 13 kidney transplant patients with squamous cell carcinoma randomized to sirolimus 21 years after transplant, although no such buildup was noted in the juxtaposition CNI group. A similar increment was noted after induction with alemtuzumab.41 Noris and associates in 2013 demonstrated that the reconstituted T-cell popu­lation after alemtuzumab induction had an exceedingly higher magnitude of regulatory T cells in patients who went through sirolimus therapy in contrast to those who received cyclosporine. From this, it can be hypothesized that a coalescence of alemtuzumab induction and maintenance therapy with sirolimus could be tolerogenic. Thus the deep-rooted feasibility of this aggrandizement stands in need of further critical appraisal. Moreover, Bansal and associates further investigated the distinctive effect of these drugs on other specifications of immunity, such as B-cell and dendritic cell function.⁴²

In general, early CNI withdrawal with subse­quent introduction of an mTOR-inhibitor-based regimen seems a more practical and realistic approach toward immunosuppressive care of renal transplant recipients. However, considering the higher rate of rejection observed in these studies, it is advisable not to offer these regimens to patients with moderate to high immunologic risk.

Mammalian target of rapamycin inhibitor-based late conversion
Late conversion from a CNI-based regimen to a combination of MMF and sirolimus has recently been investigated as a rescue strategy for patients with deteriorating renal function due to chronic allograft nephropathy. However, the limited evidence showing benefit from conversion after 6 months is not encouraging (Tables 6 and 7).

The 2009 CONVERT study conducted by Schena and associates was the first large prospective randomized clinical trial to assess safety and efficacy of converting maintenance renal allograft recipients from CNI to sirolimus at months 6-120 in patients with baseline estimated GFR > 40 mL/min. Despite increased BPAR, serious adverse events, and high dropout, the group noticed a significant improve­ment in graft function for those who remained on the conversion protocol until the end of 1 year.43 Similarly, in the 2011 ASCERTAIN trial, Holdaas and associates found no overall advantage for renal function (modified GFR) after switch from cyclosporine to everolimus in kidney transplant patients a mean of 5 years after transplant with the exception of a few patients with estimated GFR > 55 mL/min, who showed improved modified GFR.44 The outcomes reported in the above-mentioned studies indicate that, with early conversion to sirolimus-based immunosuppression from CNI, patients who receive long duration of CNI-based immunosuppression are more prone to severe renal allograft injuries and are less tolerant to the immunosuppressive changes.

Conclusions

Calcineurin inhibitors are considered a mainstay in maintenance immunosuppressive therapy. Tacrolimus is the most commonly used CNI agent. Because CNIs are associated with lower acute rejection rates, long-term graft survival has been difficult to achieve owing to nephrotoxicity that arises with chronic CNI use.45 To avoid nephrotoxicity, CNI-sparing/withdrawal strategies are initiated early after transplant by using highly efficient nonnephrotoxic immunosuppressive drugs. The most notable agents in this group are mTOR inhibitors, including everolimus and sirolimus, because of their complementary mechanism of action and favorable nephrotoxicity profile, which has opened the way for CNI reduction/withdrawal in the early posttransplant period.⁴

Clinical data on the use of everolimus and sirolimus in tacrolimus minimization strategies in renal transplant are limited. The present evidence suggests that treatment with mTOR inhibitors allows early and substantial CNI minimization. The use of sirolimus with low-dose tacrolimus has been shown to be equally efficacious, less nephrotoxic, and safer than sirolimus therapy with standard tacrolimus. However, the observed rejection rate in these studies is high; thus, it is advisable to not offer such therapies to patients with moderate to high immunologic risk. This approach has been shown to be inferior to other regimens such as MMF/tacrolimus regarding BPAR and patient/graft survival and inferior to MMF/tacrolimus and sirolimus/MMF regarding renal function. Furthermore, several studies have reported evidence that conversion to mTOR inhibitors within 6 months of transplant is beneficial, although evidence for its use after 6 months is limited. Thus, long-term trials are needed to develop optimal regimens, timing, and patient selection criteria for efficient conversion from CNI to everolimus or sirolimus while considering the paramount endpoint of graft survival.

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