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Sirolimus in solid organ transplantation: current therapies and new frontiers Sirolimus (SRL) is a mammalian target of rapamycin inhibitor, which provides an immunosuppressive effect by inhibiting cell cycle progression. The encouraging results of combined SRL–cyclosporine therapy paved the way to further immunosuppressant combinations. Although SRL is relatively non-nephrotoxic when administered as monotherapy, it pharmacodynamically enhances the toxicity of calcineurin inhibitors. Other side effects may include hyperlipidemia and myelosuppression and less commonly wound healing impairment, proteinuria, edema and pneumonitis. Surprisingly, SRL also showed encouraging properties as an antiatherogenic and antineoplastic, opening a large spectrum of new potential applications. Whether SRL can be used safely over the long term with low doses of calcineurin inhibitors requires further study. The use of SRL as a corticosteroid-sparing agent also remains to be proven in controlled trials. KEYWORDS: cancer n heart transplantation n kidney transplantation n lung transplantation n lymphocele n pediatric transplantation n pneumonitis n proteinuria n sirolimus n transplantation
Sirolimus (SRL) was isolated in the Ayerst Laboratories in Montreal, Canada, in the early 1970s from a fungus, Streptomyces hygroscopicus, found in a soil sample collected on Rapa Nui (Easter Island), hence the original name rapamycin. Although it was initially considered as a potent anticandidal antibiotic, its development was curtailed when it was found to have immunosuppressive side-effect action [1,2] . SRL blocks mesenchymal cell response to growth factors, smooth muscle cell proliferation and intimal thickening. SRL binds to intracellular protein FKBP12, thus blocking the activation of the cell cycle-specific kinase target of rapamycin (TOR). Inactivation of TOR by SRL results in the blockage of cell cycle progression at the juncture of G1 and S phase [3] . Because apoptosis is independent of TOR activity, SRL, unlike calcineurin inhibitors (CNIs), preserves apoptosis of T cells. SRL was initially approved in combination with corticosteroids and cyclosporine (CS) for prevention of rejection after renal transplantation by the US FDA in 1999. Since then, SRL has been used effectively for primary immunosuppression after organ transplantation. SRL is rapidly absorbed in the intestines, but systemic availability is lower than 30%. The terminal half-life of SRL is 62 h in adult renal transplant recipients and its steady state is usually reached within 7–14 days, which makes once-daily dosing possible. It is metabolized by liver and intestinal cytochrome P450 enzyme CYP3A4 and its metabolites are eliminated
by the GI tract (91%) and kidneys (2%). SRL whole-blood concentrations in adults are linear and dose-proportional between 3 and 12 mg/m 2 [4] . Cytochrome inhibitors such as nondihydropyridine calcium channel antagonists (e.g., diltiazem and verapamil), azole antifungals, clarithromycin and erythromycin can increase SRL concentrations and should be used only with careful drug monitoring. Enzyme inducers such as rifampicin, pheny toin and carbamazepine can markedly reduce serum SRL concentrations. Combination of CS microemulsion formulations with SRL increases exposure of both drugs when given in combination [5] , leading to a recommendation that the drugs be administered 4 h apart. This may result from competition for the intestinal p-glycoprotein counter transporter between CS and SRL or by competitive suppression of CYP3A. Because tacrolimus (TAC) is administered in dosages 100‑fold lower than CS, the drug does not share this specific interaction and SRL concentrations are similar either with simultaneous administration with TAC or with dose separation [6] .
10.2217/IMT.11.143 © 2011 Future Medicine Ltd
Immunotherapy (2011) 3(12), 1487–1497
Massimiliano Veroux*1, Tiziano Tallarita1,2, Daniela Corona1, Antonino D’Assoro3, Carmelina Gurrieri1 & Pierfrancesco Veroux1 Vascular Surgery & Organ Transplant Unit, Department of Surgery, Transplantation & Advanced Technologies, University Hospital of Catania, Via Santa Sofia, 83 95128 Catania, Italy 2 Division of Vascular & Endovascular Surgery, Mayo Clinic, Rochester, MN, USA 3 Department of Biochemistry & Molecular Biology, Mayo Clinic School of Medicine, Rochester, MN, USA *Author for correspondence: Tel.: +39 095 378 2206 Fax: +39 095 378 2358
[email protected] 1
SRL & kidney transplantation De novo SRL-based immunosuppression without CNIs CNIs have been the cornerstone of maintenance immunosuppression in kidney transplantation. However, it is well known that prolonged use of CNIs may be detrimental to ISSN 1750-743X
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long-term graft function and survival, owing to chronic nephrotoxicity, with irreversible renal dysfunction and shortened renal transplant survival. Although TAC seems less nephrotoxic than CS, a prospective randomized trial comparing low doses of CS with TAC failed to find any differences in renal scarring or function 12 months after transplantation [7] . To avoid progressive nephrotoxicity, CNI elimination or reduction has been a challenging goal after kidney transplantation. An appealing alternative was substituting SRL for a CNI, to limit the risk of renal function impairment. In early clinical trials of CNI-free SRL-based immunosuppressive protocols, a significantly better renal function was reported 1 year after the transplantation, but with an unacceptable high rate of acute rejection episodes (30–40%) [8] . To couple the possibility of a CNI-free immunosuppression with a lower acute rejection rate, many studies with a combination of the IL‑2R blocker basiliximab followed by SRL, mycophenolate mofetil (MMF) and steroids were developed, demonstrating a better renal function, lower renal fibrosis and a 10–15% acute rejection rate [9,10] . During the past years, many clinical trials have been developed, using a regimen of antibody induction followed by SRL, MMF and steroids [8–12] . Patients were highly selected (first transplant, low immunological risk, kidney only transplant) and all these trials observed biopsy-proven acute rejection (BPAR) rates of 10–15% and an estimated glomerular filtration rate (eGFR) of 65–80 ml/min the first year after transplantation. All investigators, however, highlighted the importance of therapeutic drug monitoring of SRL, in order to maintain adherence to C0 levels of at least 10–15 ng/ml. Starting from these observations, Flechner et al. suggested that CNI-free, SRLbased immunosuppression should be proposed to low- or moderate-immunological-risk, nonobese patients, whose native kidney diseases are not associated with heavy proteinuria (focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis and so forth) [8] . Several studies have compared SRL versus CNI as an initial main immunosuppressive drug with heterogeneous results [9] . In the majority of the case–control studies [9,13,14] , SRL seemed to improve graft and patient survival compared with CNI therapy, with lower incidence of acute rejection. The longer graft survival can be explained by the fact that the use of SRL avoids nephrotoxicity typically induced by CNIs, in this way prolonging graft and patient survival. 1488
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Glotz et al. found similar outcomes between SRL- and CNI-based therapy, with 95.8 versus 97.1% patient survival, with a BPAR rate of 16.9 versus 12.9% in SRL and CNI groups, respectively, and slightly higher rates of graft loss in the CNI group [15] . In the SYMPHONY study, one arm of 399 kidney transplant recipients was treated with induction therapy with daclizumab, MMF 2 g daily, steroids and low-dose SRL (C0 level targeted at 4–8 ng/ml) [16] . These patients experienced acute rejection rates of 37% at 1 year and eGFR values the same or worse than those treated with low-dose CNI-based immunosuppression. However, these results were predictable owing to the subtherapeutic target levels used in the SRL arm. More recently, the ORION study compared two SRL-based regimens (SRL + TAC, week 13 TAC elimination or SRL + MMF) with a TAC/ MMF regimen. The SRL/MMF arm was prematurely terminated owing to the higher-thanexpected BPAR rate at 2 years (32.8%) and graft and patient survival and graft functionality were similar between groups. The conclusion of the study was that SRL-based regimens were not associated with improved outcomes after kidney transplantation [17] . SRL & minimization of CNI
To reduce the risk of acute rejection and to extend the use of SRL to nonideal kidney transplant recipients, SRL was associated with low doses of CNI, by coupling the more potent immunosuppressive effect of CNI and the lower nephrotoxicity of SRL. Recent studies demonstrated that the combination between CS and SRL followed by CS minimization may be a valid alternative to CS at normal dosages by reducing the complications related to drug toxicity and either maintaining or improving graft and patient survival [18,19] . In a multicenter study conducted by Gonwa et al., living- and deceased-donor kidney transplant recipients were randomized to receive maintenance therapy with corticosteroids and either TAC and SRL (n = 185) or TAC and MMF (n = 176) [20] . After 6 months of followup, there were no differences in the incidence of acute rejection (13.0 vs 11.4%) and in overall patient and graft survival. However, early and 12‑month follow-up of renal function estimated by serum creatinine levels was significantly worse in the SRL arm. Mendez et al. demonstrated that, although overall patient and graft survival were not different, 1‑year graft survival was significantly better among patients in the future science group
Sirolimus in solid organ transplantation: current therapies & new frontiers
MMF arm when the analysis was limited to patients without delayed graft function (99 vs 93%; p = 0.01) [21] . SRL plus TAC might prevent acute rejection when compared with CS/SRL or TAC plus MMF, but they enhanced the nephrotoxicity induced by CNI [22] . However, in an analysis of almost 45,000 kidney transplant recipients from the Scientific Renal Transplant Registry [23] , CS/SRL or CS/ MMF regimens were associated with better results than TAC/SRL therapy, with improved graft survival at 3 years. However, by 24 months post-transplant, more than 55% of patients initially treated with SRL-based immunosuppression had been converted to a different regimen [23] . In summary, when used in combination with CNI, SRL therapy was not associated with increased patient and graft survival. Risk of acute rejection increases with lower doses of SRL, while lower doses of CNI may result in a better renal functionality, as expressed by improved eGFR 1 year after transplantation [24] . Conversion from CNIs to SRL
When SRL is used to replace a CNI-based immunosuppression, the critical point is the first phase of overlapping where too high a dosage can lead to high risk of nephrotoxicity or infections. Several studies have reported results from either early conversion (within the first 6 months after the transplantation) or late conversion (at least 6 months after transplantation). Weir and colleagues reported their experience of over 290 patients randomized to maintain MMF and a CNI or MMF plus SRL [13] . After 1 year, GFR was significantly higher in the MMF/SRL group compared with the MMF/CNI group. This difference was nullified at 2‑year follow-up, although MMF/CNI carried a higher mortality rate. In a recent study, 192 patients treated with induction with daclizumab and CS, MMF and steroids were randomized at 3 months to be converted to SRL or to continue CS [25] . At 1 year, creatinine clearance was significantly better in the SRL group, although the mean difference was only 4 ml/min. Patient and graft survival were not different. However, the risk of rejection was doubled in the SRL group (17 vs 8%). These findings were confirmed by a more recent study by Guba et al. [26] , who showed significantly better graft functionality in kidney transplant recipients converted to SRL regimens 10–24 days after transplant compared with CNI future science group
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maintenance regimens. However, drug discontinuation was significantly higher among SRL patients (36.2 vs 19.7%), with a higher incidence of side effects among SRL group. More recently, the postconcept study demonstrated that conversion from CS to SRL 3 months after transplantation may significantly improve renal function up to 48 months after transplantation [27] . Interestingly, a recent analysis of post-transplant biopsies in 15 patients receiving maintenance CS/SRL immunosuppression or CS withdrawal at 3 months demonstrated that CS prevents glomerular adaptation after transplantation and this may contribute to explaining why eGFR was superior in the CS withdrawal group [28] . Late CNI withdrawal appears to be safe and associated with better outcome when compared with ‘standard’ therapy, because it may circumvent the complications related to CNI use. However, studies are often not comparable owing to the lack of standardized therapy and patient clinical risk mismatching and, therefore, final conclusions cannot be easily drawn. Opelz and Dohler found conversion to SRL unsafe and associated with higher rate of graft failure when compared with the control group [29] . High rate of BPAR was also found in the CONVERT trial, where 830 transplant patients were randomized in a 2:1 ratio to either abrupt CNI withdrawal and SRL initiation or to CNI maintenance, within 6–120 months post-transplant [30] . In this trial, the intention-to-treat analysis found no difference in GFR, graft loss or death. However, in a post hoc analysis, the investigator showed that patients with a GFR 40 ml/min and a urinary protein-to-creatinine ratio of ≤0.11 showed a better graft functionality at 12 months. SRL conversion was associated with more side effects such as proteinuria, but on the other hand, the rate of malignancy was significantly lowered by SRL-based therapy. In summary, conversion from CNI- to SRLbased immunosuppression may be performed safely, without a higher risk of acute rejection but also no benefit in graft functionality. The potential long-term benefit of fewer www.futuremedicine.com
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malignancies could be a strong argument for a conversion, but should be fully evaluated by other studies. Steroid-free immunosuppression & SRL
An appealing immunosuppressive regimen may be to couple corticosteroid-free and SRL-based immunosuppression. Most of the studies of this are uncontrolled trials and among these, there are several experimental efforts to use T‑celldepleting antibodies to facilitate minimization of corticosteroids and other regimens with SRL-based maintenance therapy [31–34] . More recently, a prospective study among 45 livingdonor transplant recipients receiving TAC/SRL and steroids, randomized to undergo steroid or TAC withdrawal 3 months after transplantation, found no differences in the incidence of BPAR, graft and patient survival or in renal function between the groups, although a higher rate of discontinuation (21.7%) was observed in the TAC withdrawal group [35] . In summary, these findings suggested that conversion to a steroid-free immunosuppressive regimen can be performed safely in both lowand high-risk patients in experienced centers; nevertheless, a standardized protocol has to be developed in order to reduce the incidence of acute rejection and adverse events.
SRL & pancreas–kidney transplantation Immunosuppressive therapy with SRL has been shown to reduce the incidence of chronic allograft nephropathy related to CNI toxicity and this rationale could be even more valid in kidney–pancreas transplant recipients. In fact, in several series, SRL-based therapy resulted in lowering rejection rates in patients undergoing kidney–pancreas transplantation compared with the CNI/MMF/steroid therapy [36,37] . Vincenti and Stock reported their experience in the treatment of kidney–pancreas recipients with a corticosteroid-sparing regimen [36] . Patients who received an induction therapy with rabbit antithymocyte globulin, followed by maintenance treatment with SRL, MMF and low-dose TAC, reported rejection rates of less than 10% for each organ. Similarly, Rogers et al. successfully converted patients who received kidney–pancreas transplantation from TAC/MMF to TAC/SRL, with a low incidence of rejection despite a reduction in TAC concentrations [38] . In their interesting study, Knight et al. found similar 2‑year results and better renal function in 22 pancreas–kidney recipients 1490
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receiving thymoglobulin induction and SRLbased immunosuppression with early withdrawal of steroids and replacement of CS with mycophenolic acid at 6 months post-transplantation, compared with 20 recipients receiving thymoglobulin followed by SRL, reduced-dose CS and prednisone [39] .
SRL & liver transplantation Despite the first discouraging experiences with SRL, owing to the high incidence of wound complications, infections and hepatic arterial thrombosis when used as de novo therapy [40,41] , nowadays there is a growing interest in the use of SRL for liver transplant recipients, particularly in patients with previous hepatocellular carcinoma (HCC) or with renal function impairment. SRL exhibited antiproliferative effects against HCC in vitro and may have potential benefits in liver transplant recipients with HCC [42] . Zimmerman et al. reported on 97 patients with HCC who underwent liver transplants, who were randomized to SRL/CNI immunosuppression (45 patients) or CNI/mycophenolic acid and corticosteroids (52 patients) [43] . The 5‑year survival for patients treated with SRL was significantly higher compared with CNI-based im munosuppressed patients (78 vs 62%, respectively). In order to avoid the nephrotoxicity of CNI, SRL is been using as an adjunct or alternative immunosuppressant drug with excellent patient and graft survival, as shown by several studies [44– 46] . However, most of the studies evaluating the benefit of a conversion from CNI to SRL therapy failed to demonstrate a significant improvement in renal function and displayed an increased risk of infection, mouth ulcers and treatment discontinuation [40] . In a recent meta-analysis, Asrani et al. reported that conversion to SRL from a CNI-based immunosuppression was not associated with a significant improvement in renal function in liver transplant recipients with renal insufficiency [47] . Nevertheless, this analysis was limited by the heterogeneity of presented data. More recently, SRL monotherapy has been introduced in liver transplant recipients with HIV infection with several significant benefits in long-term immunosuppression maintenance and HIV‑1 control [48] . SRL & heart transplantation The role of mTOR inhibitors in heart transplant recipients has been evolving during last decade. SRL and its analog everolimus have been shown to slow the progression of cardiac allograft vasculopathy (CAV) [49–51] . future science group
Sirolimus in solid organ transplantation: current therapies & new frontiers
In one of the earlier, randomized, open-label studies, SRL was compared with azathioprine in combination with CS and steroids administered from the time of cardiac transplantation, to evaluate the 6‑month rejection rates and the 6‑ and 24‑month graft vasculopathy [50] . SRL-based immunosuppression displayed a reduced incidence of acute rejection and a highly significant absence of progression in intimal plus medial proliferation and significant protection against luminal encroachment at 2 years compared with patients treated with azathioprine. The mechanism by which SRL prevents vasculopathy includes regulation of proliferation and migration of vascular smooth muscle cells, heightened production of nitric oxide, antiangiogenesis effects and inhibition of extracellular matrix accumulation and fibrosis [50] . Moreover, the progression of CAV may be reduced after conversion from CNI to SRL [52] . Raiclin et al. observed that, in heart transplant recipients treated with SRL, there is a less ��������������� pronounced coronary epicardial endothelial dysfunction compared with CS, possibly reducing the incidence and progression of CAV [51] . Delgado et al. collected efficacy and safety data from a Spanish registry of heart transplant recipients who were switched from a CNI to SRL owing to renal failure [53] . A total of 97 patients started SRL owing to renal impairment. When SRL was started, CNIs were progressively tapered and in some cases withdrawn. Only one episode of acute rejection was observed in a patient receiving SRL plus CS but the eventual allograft function remained stable [53] . A recent study, however, demonstrated that TAC/MMF combination was associated with less acute rejection episodes and an improved side-effect profile compared with TAC/SRL combinations [54] . More recently, conversion to SRL plus dosereduced CNI was associated with a stabilization of renal function in heart transplant recipients [55,56] .
Lung transplantation SRL as a primary immunosuppressant in lung transplantation has been burdened by high incidence of postoperative airway dehiscence and consequent early deaths. Better outcome has been seen with SRL in the late conversion (with improvement of renal function and no major pulmonary complications) and in combination with low doses of CNI. In particular, SRL plus low dosages of CNI was associated with improvement of bronchiolitis obliterans in 30% of patients in a small series [57] . future science group
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Pediatric transplantation There are few studies analyzing the role of SRL as part of the immunosuppressant therapy in children. A group from Spain used SRL for primary immunosuppression along with MMF, steroids and two doses of basiliximab in six children receiving their first kidney transplants. Patients also received CS, which was withdrawn during the first week post-transplant when therapeutic levels of SRL were achieved (4–12 ng/ml). There were no rejection episodes during 1‑year followup and SRL was well tolerated without adverse effects [58] . De novo use of SRL or conversion from a CNI to SRL may constitute a viable option to reduce or to avoid CNI-induced nephrotoxicity in pediatric renal transplant recipients. However, complete CNI-free immunosuppression in pediatric recipients seems to be inappropriate, because it often requires lymphocyte-depleting antibodies for rejection prophylaxis. Some studies demonstrated a slight improvement of renal function in pediatric kidney transplant recipients, but this was attained at the cost of a higher acute rejection rate in 10–20% of patients [59] . At present, SRL/ MMF and corticosteroid therapy does not appear safe in pediatric renal transplant recipients since it is associated with an increased risk of acute rejection episodes [59] and SRL, combined with lowdose TAC, was not effective at mitigating progressive histological changes or functional decline associated with chronic renal allograft injury [60] . SRL-based therapy has been used successfully in nonrenal pediatric organ transplant recipients, usually to lower CNI dosage and prevent or overcome complications related to the use of CNI drugs [61,62] . However, recent studies confirmed that the use of SRL in pediatric recipients is not safe: McDonald et al. studied 274 pediatric renal transplant recipients receiving basiliximab, CNI, SRL and steroids, who were randomized to steroid withdrawal or minimization, and demonstrated an unacceptable higher rate of posttransplant lymphoproliferative disorders among SRL-treated patients, without a clear benefit in terms of graft function [63] . Although this combination permitted a safe steroid withdrawal in 73 patients, the use of SRL/CNI combined immunosuppression in pediatric renal transplant recipients is not recommended [64] . SRL & cancer One of the most interesting properties of mTOR inhibitors is to reduce TGF and VEGF, involved in tumor growth, consequently resulting in www.futuremedicine.com
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antitumoral effects. In long-term studies, SRL-based immunosuppression therapy was associated with a relatively low incidence of malignancy compared with other immuno suppressants. SRL alone or in combination with CS prevented metastatic tumor progression and prolonged survival in mice inoculated with renal cancer cells. Interestingly, mTOR-dependent signaling within endothelial cells is induced by VEGF and plays a key role in the angiogenic effects of this growth factor. Indeed, mTOR inhibitors have been shown in vitro and in vivo to be potent blockers of angiogenesis. This peculiar characteristic of SRL may explain its potential role both in vascular modification occurring in chronic allograft nephropathy and malignancy growth [65] . This effect was seen to have clinical relevance, since conversion to SRL was able to induce a complete remission of cutaneous Kaposi’s sarcoma in a series of 15 cases [66] . Data from the United Network for Organ Sharing Registry confirmed that patients treated with SRL therapy had a lower incidence of skin and solid organ cancer than CNI-treated patients [67] . Campistol et al. showed that, at 5 years posttransplantation, the incidence of any type of tumor was significantly lower in patients treated with SRL and steroids than in those treated with SRL plus CS and steroids [68] . This observation was further confirmed by the CONVERT trial, which demonstrated that, after conversion to SRL therapy, the incidence of cancer was three‑times lower than in those with CNI continuation [30] .
Management of side effects of SRL Hyperlipidemia Hyperlipidemia is the most frequent SRL side effect [69] . Several experiences have shown a dose-dependent decrease in the catabolism of ApoB100‑containing lipoproteins, resulting in increasing of the lipid profile [70,71] . Most studies demonstrated that this effect is reversible and hyperlipidemia can be successfully controlled by statins and fibrates [69,72] . However, since both SRL and lipid lowering are metabolized by the CYP3A, the incidence of adverse effects of both drugs can be increased [73] . Despite worsening of the lipidemia, SRL protects from the development of atherosclerosis by inhibiting vascular smooth cell proliferation. As shown above, this interesting effect can prevent CAV in organs such as the heart and kidney, playing a protective role on the allograft. 1492
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To date, there are no clinical data to determine the effects of mTOR inhibitor dyslipidemia on cardiovascular disease in kidney transplant recipients. Myelosuppression Thrombocytopenia remains one of the major and most common side effects of SRL. This appears to be influenced by drug concentration; therefore, patients can experience relief from the adverse affect by lowering the dosage or withdrawing SRL [74] . The mTOR inhibitors besides reduce the effect of several growth factors including erythropoietin, thrombopoietin and IL‑3, consequently leading to anemia as well. In a recent review, SRL was one of the most effective risk factors in inducing anemia in renal transplant recipients receiving TAC plus SRL or TAC plus MMF. In particular, the prevalence of anemia was 57% in the SRL group versus 31% in MMF group (p
