Sirolimus and its closely related analog everolimus are potent immunosuppressive agents that impair lymphocyte activation and proliferation by inhibiting the mammalian target of rapamycin (mTOR), also known as the mechanistic target of rapamycin. The mTOR inhibitors emerged in the 1990s as a new class of immunosuppressive agent and a promising alternative to calcineurin inhibitor (CNI)-based therapy in organ transplantation, particularly because mTOR inhibitors were not believed to cause nephrotoxicity, hitherto the Achilles’ heel of CNIs. Their appeal was further enhanced by the suggestion that they may have antitumor effects and inhibitory effects on vascular smooth muscle proliferation that might help prevent chronic rejection.
The early promise that mTOR inhibitors would replace CNIs as the mainstay of immunosuppression after organ transplantation has not been fulfilled. Their side effects and limitations became increasingly apparent, and as a result their use in organ transplantation has been more limited than initially anticipated. As new information from clinical trials of the mTOR inhibitors emerges it is clear they still have an important role and are an effective alternative to CNI-based immunosuppressive therapy in a number of clinical situations.
Discovery
Sirolimus (AY-22989, rapamycin, Rapamune) is a fermentation product of the bacterium Streptomyces hygroscopicus, first isolated from soil samples taken in 1965 from Easter Island, which is known locally as Rapa Nui, hence the decision to subsequently name the drug rapamycin. It was first investigated as an antifungal agent in the mid-1970s , and in 1977 it was also reported to have immunosuppressive effects in the rat, where it prevented the development of experimental allergic encephalomyelitis and adjuvant arthritis. Twelve years later it was reported that administration of rapamycin prolonged the survival of heart allografts in rats and kidney allografts in pigs and dogs, although it was noted that some pigs developed interstitial pneumonitis, an observation that was later to be repeated in the clinic. More worryingly, rapamycin resulted in lethal vasculitis of the gastrointestinal tract in dogs, and this finding delayed further clinical evaluation of sirolimus. Tacrolimus, which shares structural similarity with sirolimus ( Fig. 18.1 ), also causes vasculitis in the dog, but when used in humans in 1989 there was no sign of such toxicity, an observation that resulted in a resumption in the clinical evaluation of sirolimus.
As the potential of sirolimus as a clinical immunosuppressive agent became apparent, chemists at Novartis synthesized everolimus (RAD001, SDZRAD, Certican, Zortress, Afinitor, Votubia) by making a 2-hydroxyethyl chain substitution at position 40 of the sirolimus molecule (see Fig. 18.1 ), a drug that retained potent immunosuppressive activity with improved oral bioavailability. Subsequently more derivatives have been synthesized with the aim of harnessing its antitumor effects.
Mechanism of Action
The mTOR inhibitors, sirolimus and everolimus, exert their principal immunosuppressive effects by inhibiting the ability of the intracytoplasmic mTORC1 multiprotein enzyme complex to regulate the growth, proliferation, and survival of lymphocytes and other immunocompetent cells. mTOR (a 289kDa serine-threonine protein kinase) is a central component of two functionally distinct complexes called mTOR complex 1 and 2 (mTORC1 and mTORC2; Fig. 18.2 ). Both mTOR complexes exert their effects on other intracellular signaling molecules including Akt and S6 kinase. Sirolimus and everolimus are potent inhibitors of mTORC1, whereas the mTORC2 complex is relatively resistant to these mTOR inhibitors.
After entering into cells, the mTOR inhibitors bind to one of a family of immunophilins called FK506-binding proteins (FKBPs), particularly the 12-kD FKBP12 (see Fig. 18.2 ). Immunophilins are highly conserved and abundantly expressed cytosolic proteins whose natural function in the cell is the cis/trans isomerization of peptidyl-prolyl bonds and is completely distinct from their ability to act as receptors for certain immunosuppressant molecules and mediate immunosuppressive effects. The sirolimus-FKBP12 or everolimus-FKBP12 molecular complex binds to the FKBP12-binding domain of mTOR and blocks association of Raptor (rapamycin-sensitive adapter protein) with mTOR and mLST8 and other proteins that collectively comprise the functional mTORC1 complex within the cell. This interferes with mTORC1-dependent intracellular signaling pathways, particularly those regulating cell growth and proliferation in response to signals from cytokines, growth factors, nutrients (especially amino acids), stress (e.g., hypoxia) and toll-like receptor (TLR) ligand engagement. In lymphoid cells, the important signals originate from the cell surface and are generated by cytokine receptor binding, such as binding of interleukin-2 to the interleukin-2 receptor complex, and ligand binding to coreceptors such as CD28.
The mTORC2 complex comprises mTOR, Rictor (rapamycin insensitive companion of mTOR), mitogen-activated protein kinase associated protein (mSIN1), protein observed with Rictor (Protor/PRR5), and mLST8. Whereas mTORC2 is resistant to acute inhibition by sirolimus (and everolimus), increasing evidence suggests that chronic exposure to sirolimus may block mTORC2 function. mTORC2 is involved in regulating cell morphology and maintaining the integrity of the cytoskeleton. It is also an important regulator of intracellular signaling pathways in both B and T cells, and mTORC2 inhibition may contribute to immunosuppressive effects of sirolimus and everolimus.
Functionally distinct T cell subsets exhibit differential susceptibility to mTOR inhibition and, interestingly, mTOR inhibitors appear to preferentially promote the expansion of T regulatory cells in humans, which makes them attractive agents to include in experimental protocols aimed at promoting transplant tolerance. The extent to which this property of mTOR inhibitors contributes to their clinical effectiveness as immunosuppressive agents remains to be defined.
Whereas the principal immunosuppressive effect of mTOR inhibitors has been attributed to their inhibitory effect on T cell proliferation, with activated lymphocytes showing cell cycle arrest in the late G1 phase, it is now clear that mTOR functions as a master regulator controlling many aspects of both innate and adaptive immunity. The mechanisms of immunosuppression through mTOR inhibition are diverse, complex, and still not fully understood. For example, mTOR regulates T cell trafficking through altered expression of cell surface molecules such as CCR7, and adhesion molecules such as VCAM1 on endothelial cells. Therefore mTOR inhibition can alter lymphocyte migration patterns significantly. mTOR plays an important role in regulating the differentiation of functionally distinct CD4 + T cell subsets and mTOR blockade appears as noted previously to preferentially promote the development of T regulatory cells. mTOR also influences B cell development and maturation, and inhibiting mTOR may interfere with B cell responses. Inhibition of mTOR has been shown to have both immunosuppressive and immunostimulatory effects on dendritic cells, reducing and increasing their ability to stimulate T cells according to the type of dendritic cell. Paradoxically, inhibiting mTOR signaling may also have immunostimulatory effects on T cells, and sirolimus may, for example, promote the generation of long-lived memory T cells.
Pharmacokinetics
Sirolimus and everolimus are only available as oral formulations, and both are marketed in tablet form; sirolimus is also available as an oral solution. They are rapidly absorbed from the intestine although both have a relatively low and variable bioavailability (around 25%). Both sirolimus and everolimus have a narrow therapeutic index and therapeutic monitoring of blood levels is necessary to ensure effective and safe immunosuppression. Glutathione-everolimus, an injectable prodrug of everolimus, has been developed, largely on the back of everolimus in the oncology field.
After absorption, sirolimus is extensively bound to erythrocytes, and less than 5% of the drug remains free in the plasma, where it is associated with the nonlipoprotein fraction. It has a long half-life of about 60 hours in renal transplant patients, with rapid absorption time to maximal concentration at 1 to 2 hours and exposure that is proportional to dose, but with a large intersubject coefficient of variance (CV; CV = 52%) and significant intrasubject variability (CV = 26%). The pharmacokinetic profiles of the tablet and liquid formulations of sirolimus are similar apart from a lower maximal concentration with tablets. With both formulations, the total drug exposure (area under the concentration-time curve [AUC]) correlates well with maximal and trough concentrations. Similar to cyclosporine and tacrolimus, the pharmacokinetics of sirolimus differ in different ethnic groups, with reduced oral bioavailability in African Americans.
Everolimus is more water-soluble than sirolimus, and this increases its bioavailability. In studies of single doses of everolimus capsules in renal transplant recipients, everolimus was shown to have a much shorter half-life than sirolimus (16–19 hours), a rapid absorption (maximal concentration reached within 3 hours), and a good correlation between trough and AUC. As with sirolimus, ethnicity affects everolimus pharmacokinetics, with a higher dose requirement in African American patients. There is a difference in the bioavailability of everolimus depending on the size of tablet taken, with a higher C max when five 1 mg tablets are taken compared with a single 5 mg tablet.
Therapeutic Blood Monitoring
The low oral bioavailability of mTORs, effects of concomitant food and other medicines, and variations in the patient’s condition may cause significant inter- and intrapatient variation in whole blood concentrations of mTORs. The requirement to optimize exposure to the drugs to maintain adequate immunosuppressive efficacy while minimizing side effects mandates monitoring of whole blood concentrations of the drug, and militates against fixed-dose regimens. Whole blood measurements of drug concentration are performed because of the high degree of binding of the drug to erythrocytes, although it is the very small free drug fraction that is responsible for immunosuppression. There are two principal methods used in the determination of mTOR concentrations, enzyme-linked immunoassays and high-performance liquid chromatography (HPLC) with ultraviolet or mass spectrometry detection. HPLC measures the parent drug; it is very accurate but time consuming. Immunoassays, on the other hand, use microparticles coated with antibodies to the mTORs and provide a rapid assay, but one which overestimates the drug concentration because of cross-reaction with drug metabolites in addition to the parent drug. Even with the same assay there may be variations in measured concentrations between laboratories and over time.
Pharmacogenetics
Sirolimus and everolimus are metabolized in the liver and intestinal wall by the cytochrome P450 (CYP) 3A enzyme subfamily (CYP3A4 and CYP3A5) and to a minor extent by CYP2C8. Polymorphisms of these enzymes are common and because of linkage disequilibrium (the genes lie adjacent on chromosome 7q21) may occur together. Polymorphisms of CYP3A enzymes are associated with lower drug concentration-to-dose ratios in patients expressing the least common genotypes. An association between CYP3A5 genotype and dose requirement for sirolimus has been noted in some but not all studies, and a recent study in renal transplant patients found no association between CYP3A5 genotype and everolimus pharmacokinetics. It is likely that CYP3A5 contributes much less to sirolimus metabolism than CYP3A4, and that many of the studies of the CYP3A5 interaction have been underpowered.
Drug Interactions
Because sirolimus and everolimus are metabolized primarily by CYP3A4, and to a lesser extent by CYP3A5 and CYP 2C8, drugs that affect these enzyme pathways alter the metabolism of the mTORs. Important among these are the CNIs, particularly cyclosporine, which can increase the concentration of mTOR inhibitors with a reciprocal increase in cyclosporine concentrations. This drug interaction is particularly noticeable when the time interval between mTOR inhibitor and cyclosporine ingestion varies. It is thus important that patients receiving mTOR inhibitors and cyclosporine adhere to a regular pattern of medication and do not vary the interval between taking the two agents. Other data suggest that higher doses of everolimus are required to achieve the same drug exposure when coadministered with tacrolimus compared with cyclosporine. Conversely, mTOR inhibitors reduce the exposure to tacrolimus when the two drugs are coadministered. Other groups of drugs with important interactions with the CYP pathway are antimicrobials (especially fluconazole and erythromycin) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA) inhibitors (statins), both of which are widely used in renal transplant recipients.
mTOR inhibitors also differ from cyclosporine in the way they interact with other immunosuppressive agents. Patients taking sirolimus, like tacrolimus, have a much higher exposure to mycophenolic acid than do patients taking mycophenolate and cyclosporine, and for patients switching from a CNI to mTOR inhibitor the dose of mycophenolic acid may need to be adjusted. Administration of mTOR inhibitors may also result in a modest reduction in exposure (reduced AUC) to prednisolone compared with cyclosporine.
Use of mTOR Inhibitors
mTOR inhibitors have been evaluated for use in renal transplantation as an addition to CNI-based therapy and as a substitute for CNIs. mTOR inhibitors have also been used as de novo treatment from the time of renal transplantation, as a later addition to CNIs to enhance immunosuppression in response to acute rejection, and as a substitute for CNIs to avoid CNI toxicity in the maintenance phase of immunosuppression.
Early in vitro and in vivo studies suggested that mTOR inhibitors and CNIs when used together had a synergistic immunosuppressive effect. This might be anticipated given that CNIs and mTOR inhibitors each block different signals during T cell activation and affect different stages of the cell cycle. Initially, it was envisaged that mTOR inhibitors might be best used along with CNIs to exploit this synergistic immunosuppressive effect, optimizing immunosuppression and minimizing agent-specific side effects. Evidence from rodent studies suggested, however, that sirolimus may exacerbate cyclosporine nephrotoxicity, a finding that was subsequently confirmed in clinical studies. Most of the initial work with sirolimus was done in conjunction with cyclosporine rather than tacrolimus because it was believed that competition for binding to the immunophilin FKBP12 would preclude coadministration of tacrolimus and sirolimus. It later became evident that there is an abundance of FKBP12 in the cytoplasm, and in vitro studies suggest that less than 5% of the available FKBP needs to be bound to cause half-maximal immunosuppression. Tacrolimus and sirolimus can be administered simultaneously at therapeutic doses in humans without significant competition for FKBP12.
De Novo Therapy with mTOR Inhibitors in the Absence of Calcineurin Inhibitors
Sirolimus has been investigated in numerous studies where it was the principal immunosuppressant. The first such studies were phase 2 trials conducted in Europe that examined concentration-controlled sirolimus dose regimens, rather than fixed-dose regimens. When sirolimus was administered as a component of triple therapy with azathioprine and prednisolone, it was associated with a similar incidence of acute rejection to that observed in patients on the old nonmicroemulsion Sandimmune preparation of cyclosporine (41% vs. 38% at 12 months). A follow-up study substituted azathioprine with mycophenolate mofetil (MMF) and showed no significant difference in the incidence of acute rejection between sirolimus and cyclosporine, although there were numerically more acute rejection episodes in the sirolimus arm (27.5% vs. 18.5%). Patient and graft survival were similar in the two study groups, although the studies were insufficiently powered to detect small differences. Pooled data from both studies showed significantly better renal function in patients receiving sirolimus. These two early studies provided the first detailed insight into the toxicity profile of sirolimus in humans and suggested a side effect profile that was different from that associated with CNIs ( Table 18.1 ).
| Sirolimus ( n = 41 + 40) | Cyclosporine + Azathioprine ( n = 42) | Cyclosporine + MMF ( n = 38) | |
|---|---|---|---|
| Metabolic | |||
| Hypertriglyceridemia | 21 + 29 = 50 (63%) | 5 (12%) | 19 (50%) |
| Hypercholesterolemia | 18 + 26 = 44 (54%) | 6 (14%) | 17 (45) |
| Hyperglycemia | 8 + 6 = 14 (17%) | 3 (7%) | 6 (16%) |
| IDDM | 1 + 1 = 2 (2%) | 1 (2%) | 1 (3%) |
| ALT increase | 8 + 8 = 16 (20%) | 1 (2%) | 3 (8%) |
| Hypokalemia | 14 + 8 = 22 (27%) | 0 | 6 (16%) |
| Hypophosphatemia | 6 + 6 = 12 (15%) | 0 | 1 (3%) |
| Hyperuricemia | 1 (3%) | — | 7 (18%) |
| Hematologic | |||
| Thrombocytopenia | 15 + 18 = 33 (41%) | 0 | 3 (8%) |
| Leukopenia | 16 + 11 = 27 (33%) | 6 (14%) | 7 (18%) |
| Anemia | 15 + 17 = 32 (40%) | 10 (24%) | 11 (29%) |
| Infections | |||
| CMV viremia | 6 + 2 = 8 (10%) | 5 (12%) | 8 (21%) |
| Herpes simplex | 10 + 6 = 16 (20%) | 4 (10%) | 6 (16%) |
| Herpes zoster | 0 + 1 = 1 (1%) | 1 (2%) | 1 (3%) |
| Oral Candida | 3 + 5 = 8 (10%) | 0 | 3 (8%) |
| PCP | 0 + 0 | 1 (2%) | 0 |
| Pyelonephritis/UTI | 17 + 17 = 34 (42%) | 12 (29%) | 15 (39%) |
| Septicemia | 6 + 2 = 8 (10%) | 1 (2%) | 1 (3%) |
| Pneumonia | 7 + 6 = 13 (16%) | 1 (2%) | 2 (5%) |
| Wound infection | 4 + 2 = 6 (7%) | 2 (5%) | 3 (8%) |
| Other | |||
| Hypertension | 7 + 16 = 23 (16%) | 14 (33%) | 18 (47%) |
| Arthralgia | 8 (20%) | 0 | — |
| Tremor | 1 + 2 = 3 (4%) | 7 (14%) | 8 (21%) |
| Gingival hyperplasia | 0 + 0 | 4 (10%) | 3 (8%) |
| Hirsutism | 1 (3%) | — | 4 (11%) |
| Diarrhea | 15 (38%) | — | 4 (11%) |
| Malignancies | 0 | 2 (5%) | 0 |
Subsequent studies further explored the use of sirolimus with MMF, together with anti-CD25 monoclonal antibody induction therapy. Early data suggested that the combination of sirolimus and MMF was superior to a cyclosporine-based regimen. A subsequent randomized trial comparing sirolimus and tacrolimus (each given along with MMF and prednisolone) showed the two regimens to be comparable in terms of acute rejection rate and graft function. A registry analysis suggested that renal allograft recipients treated with a combination of sirolimus and MMF had a higher rate of acute rejection and reduced allograft survival compared with recipients receiving alternative immunosuppressive regimens.
A systematic review of randomized trials in which mTOR inhibitors were used in place of CNIs as initial therapy after kidney transplantation in studies published up to 2005 (eight different trials with a total of 750 participants) revealed that there was no difference in the incidence of acute rejection at 1 year, but the level of serum creatinine (a possible surrogate endpoint for long-term graft survival) was lower in patients receiving mTOR inhibitors.
Reports from two large-scale trials (ORION and SYMPHONY) suggested that the combination of sirolimus and MMF is inferior to a low-dose tacrolimus and MMF-based triple therapy.
The ORION study was an open-label randomized multicenter international comparison of two sirolimus-based regimens with tacrolimus and MMF in adult first- or second-time recipients of living or deceased donor kidneys. The study recruited 469 patients in 65 centers in North America, Europe, and Australia and randomly assigned them to one of three groups. Group one received sirolimus (initial target trough levels 8–15 ng/mL) plus tacrolimus (target trough level 6–15 ng/mL), with stepwise withdrawal of tacrolimus after week 13 and continuation on sirolimus (maintenance trough levels of 12–20 ng/mL). Group two received sirolimus (initial target trough levels of 10–15 ng/mL, reducing after 13 weeks to 8–15 ng/mL and after 26 weeks to 5–15 ng/mL) plus MMF (up to 2 g/day). Group three received tacrolimus (target trough levels of 8–15 ng/mL to week 26 and 5–15 ng/mL thereafter) and MMF (up to 2 g/day). The primary endpoint of the study was glomerular filtration rate (GFR) at 12 months (using the Nankivell formula) and secondary endpoints included GFR at 2 years, patient and graft survival, and biopsy-confirmed acute rejection. Study group two (Sirolimus + MMF) was prematurely discontinued because of a higher than expected rate of acute rejection. No significant differences in the primary endpoint were found. Patient survival was similar in all three groups (2-year patient survival 94.4% in group one, 94.5% in group two, and 97% in group three). Graft loss was numerically higher in the groups receiving sirolimus but not significantly so (2-year graft survival 88.5% in group one, 89.9% in group two, and 95.4% in group three). The incidence of biopsy-proven acute rejection in the first 2 years was significantly higher in the CNI-free groups (group one 17.4%, group two 32.8%, and group three 12.3%). Adverse events were the primary reason for patients not continuing in the study, and this occurred in 34.2% patients in group one, 33.6% patients in group two, and 22.3% patients in group three. The authors concluded that sirolimus-based regimens were not associated with improved outcomes after kidney transplantation.
The Efficacy Limiting Toxicity Elimination (ELiTE)-Symphony study, was an open label multicenter international randomized four-arm trial that examined the efficacy of reduced dose sirolimus, tacrolimus, or cyclosporine when combined with CD25 monoclonal antibody induction (daclizumab), MMF, and corticosteroids in adult renal transplant recipients. Both living donor and deceased donor transplants were included. The outcomes were also compared with those in recipients given standard dose cyclosporine, MMF, and corticosteroids. The primary endpoint was estimated GFR 12 months after transplantation. The study recruited 1645 patients from 15 participating countries. The study group that was given low-dose tacrolimus, MMF, and steroids had a significantly better 12-month GFR compared with the other three study groups. Acute rejection (biopsy-proven) in the first 6 months was three times higher in the low-dose sirolimus group (35.3%) compared with the low-dose tacrolimus group (11.3%). Allograft survival at 12 months (censored for death with a functioning transplant) was also significantly lower in the low-dose sirolimus and standard dose cyclosporine groups (91.7% and 91.9%, respectively) compared with the low-dose tacrolimus group (96.4%). Withdrawal from the study, mainly because of treatment failure (use of additional immunosuppressive agents and discontinuation of the study drug), was highest in recipients randomized to low-dose sirolimus (48.9%). Serious adverse events occurred in 53.2% of patients in the low-dose sirolimus group compared with around 44% for the other study groups. In this study, therefore, low-dose sirolimus resulted in higher rates of biopsy-proven rejection and no improvement in renal function compared with the cyclosporine-based regimens.
In a subsequent report on this study, with an additional 2 years of follow-up based on around half of the patients originally recruited to the (ELiTE)-Symphony study, renal function remained relatively stable overall and the incidence of graft rejection and graft loss remained low. The low-dose tacrolimus group continued to have the highest GFR but the difference compared with the other groups was no longer significant. However, the study was not powered to detect differences in the key endpoints at 3 years. The number of patients in the low-dose sirolimus group who remained on sirolimus for the entire 3-year study period was low, although their average renal function at 3 years was numerically but not significantly slightly higher than that of patients remaining on the original agents in the other three groups.
Eight-year follow-up of the 145 patients randomized in the SPIESSER study, comparing de novo cyclosporine with de novo sirolimus, together with MMF and steroids, showed similar results to ELiTe. There were more discontinuations of sirolimus than cyclosporine (47% vs. 27%) and more adverse events in the sirolimus group, but no difference in patient or graft survival, or acute rejection incidence; intention-to-treat analysis showed that the sirolimus arm had better renal function (modification of diet in renal disease study [MDRD] GFR 62.5 mL/min sirolimus [SRL], 47.8 mL/min cyclosporine [CsA], P = 0.004).
De Novo Combination Therapy With mTOR Inhibitors and Calcineurin Inhibitors
One of the first studies of sirolimus in renal transplantation to be performed was a dose-ranging study that combined different fixed doses of sirolimus in conjunction with a high-dose or low-dose Sandimmune cyclosporine (concentration controlled). All groups received steroids but no azathioprine or MMF. Small numbers of patients in the study and an unequal distribution of African Americans between the six study groups meant that the results were difficult to interpret. Nevertheless, the study showed that the combination of sirolimus and cyclosporine was more potent than cyclosporine alone in the prevention of acute rejection and that half-dose cyclosporine and sirolimus was as efficacious as full-dose cyclosporine and sirolimus. The higher incidence of acute rejection seen in African Americans in this study was also observed in subsequent studies. The other important finding to emerge from this study was a high incidence of pneumocystis pneumonia in sirolimus-treated patients, particularly where routine prophylaxis against Pneumocystis jirovecii was not given. Two large phase 3 studies of sirolimus followed shortly afterward, one conducted in the US and the second worldwide ( Table 18.2 ). Similar to earlier studies, these studies used a fixed dose of sirolimus (2 or 5 mg/day) in combination with concentration-controlled cyclosporine. In the US study, the two different doses of sirolimus were compared with azathioprine, and all groups received steroids but no induction therapy. Only patients with functioning renal allografts were recruited, in contrast to the global study in which function of the graft was not a prerequisite for enrolment. The other major difference between the US and the global study was that the comparator in the global study was placebo rather than azathioprine. Both studies showed a clear benefit in terms of reduction in the rate of acute rejection for patients receiving sirolimus, an effect that was more marked in patients receiving a higher dose of sirolimus. There was a difference in acute rejection rates, patient survival, and graft survival in the US study compared with the global study in all treatment arms, which likely reflects the different enrollment requirements, with only recipients with functioning grafts being entered into the US study.
| US Study ( n = 719) | Global Study ( n = 576) | |||||
|---|---|---|---|---|---|---|
| Aza ( n = 161) | SRL 2 mg ( n = 284) | SRL 5 mg ( n = 274) | Placebo ( n = 130) | SRL 2 mg ( n = 227) | SRL 5 mg ( n = 219) | |
| Acute rejection (%) | 29.8 | 16.9 a | 12 b | 41.5 | 24.7 c | 19.2 d |
| Creatinine clearance (mL/min) | 68.8 | 62.3 | 59.2 | 62.6 | 56.4 | |
| Graft survival (%) | 94.4 | 94.3 | 92.7 | 87.7 | 89.9 | 90.9 |
| Patient survival (%) | 98.1 | 97.2 | 96 | 94.6 | 96.5 | 95 |
a P = 0.002 relative to the azathioprine arm.
b P < 0.001 relative to the azathioprine arm.
c P = 0.003 relative to the placebo arm.
These two pivotal studies in the development of sirolimus reveal much about how best to use sirolimus and its drawbacks. There was a high incidence of lymphocele formation (12%–15% vs. 3% in the azathioprine control group in the US study) and wound infection compared with the control arm. Of particular importance was the observation that the renal function of patients on a combination of sirolimus and cyclosporine was worse than that of patients on cyclosporine alone, with a 12-month calculated creatinine clearance of 67.5 mL/min in the azathioprine control group compared with 62 mL/min and 55.5 mL/min in the 2 mg and 5 mg sirolimus groups. A similar effect was seen in the global study.
The immunosuppressive synergy between cyclosporine and sirolimus in these studies was analyzed by median effect analysis of the pooled data. This analysis showed that administration of sirolimus permitted a 2.2-fold reduction in cyclosporine exposure, and reciprocally, cyclosporine permits a 5-fold reduction in sirolimus dose to achieve the same immunosuppressive efficacy. Experimental data also suggest that synergism accounts for the increased nephrotoxicity.
The combination of everolimus and cyclosporine has been evaluated in several multicenter clinical trials. An early multicenter clinical trial conducted predominantly in Europe compared two fixed doses of everolimus (1.5 and 3 mg/day) against MMF, with all three arms receiving corticosteroids and full dose cyclosporine with target trough levels of 150 to 400 ng/mL until week 4 and then 100 to 300 ng/mL. There was no significant difference between the three groups in the primary composite endpoint at 6 months (biopsy-proven acute rejection, graft loss, death, and loss to follow-up). Significantly worse renal function in both everolimus groups led to a protocol revision at 12 months with reduction in the target trough cyclosporine levels to 50–75 ng/mL. At 3 years, renal function was similar in the low-dose everolimus and MMF groups, but significantly worse in the higher dose everolimus group. A similar trial conducted predominantly in North America with the same composite endpoint at 36 months and the same protocol amendment showed that all three groups were similarly efficacious but creatinine clearance was lower at 12 and 36 months in the everolimus groups. Another multicenter randomized phase 2 study undertaken in the US and Europe evaluated the efficacy and safety of everolimus 3 mg/day together with basiliximab induction, prednisolone, and either full dose or reduced dose cyclosporine (trough levels 125–250 ng/mL, respectively) although a protocol amendment at 12 months reduced the cyclosporine trough target to 50–75 ng/mL in all patients. The primary endpoint was efficacy (composite of biopsy-proven acute rejection, graft loss, death, or loss to follow-up) and efficacy failure was significantly higher in the full dose cyclosporine arm at 36 months (35.8% vs. 17.2%). Creatinine clearance was similar in the two groups at 36 months.
Sirolimus has also been evaluated in combination with tacrolimus after an initial report suggesting that the theoretical misgivings about the combination are not seen in clinical practice. As noted previously, the ORION study included a group that received sirolimus plus tacrolimus but with stepwise withdrawal of tacrolimus after week 13 and continuation on sirolimus.
Sirolimus in combination with tacrolimus was compared with tacrolimus and MMF as primary immunosuppression after kidney transplantation in a randomized multicenter trial in the US. A total of 361 patients were recruited. Both arms of the study received corticosteroids and sirolimus was dosed to achieve blood levels of 4 to 12 ng/mL. At 1 year renal function was superior in the tacrolimus arm and patients receiving sirolimus had a higher incidence of study drug discontinuation (26.5% vs. 14.8%). The inferiority of sirolimus/tacrolimus in terms of renal function observed in this study was not found in a more recent European multicenter randomized phase 2 clinical trial involving 734 patients. In this study primary immunosuppression using sirolimus and tacrolimus was compared with tacrolimus and MMF. Sirolimus was given according to a fixed-dose regimen of 2 mg for 28 days and 1 mg thereafter. Both study groups received steroids initially but these were tapered and discontinued after day 90. Renal function at 6 months, the primary endpoint, was similarly good in the two study groups. Acute rejection and graft survival were similar in both groups, but premature withdrawal because of adverse events was twice as high in the sirolimus/tacrolimus arm (15.1% vs. 6.3%).
Although not comparing de novo combination of sirolimus and tacrolimus, in the ADHERE study patients were given prolonged-release tacrolimus and converted from MMF to sirolimus with low-dose tacrolimus early posttransplant, on day 28. Of 730 patients randomized, 625 patients completed the study 11 months later. There was no difference in the primary endpoint of iohexol-measured GFR at 12 months (40.73 MMF vs. 41.75 mL/min/1.73m 2 SRL, P = 0.405), but there were more patients withdrawing from the sirolimus arm because of adverse events than the MMF arm (14.4% vs. 5.2%).
Everolimus plus tacrolimus has also been evaluated for use as primary immunosuppression after renal transplantation. The first prospective study to evaluate this combination was a 6-month multicenter study performed in the US. Ninety-two de novo renal transplant recipients were randomized to receive everolimus, steroids, and basiliximab together with either standard or low-exposure tacrolimus. The differences in tacrolimus exposure achieved in the two study arms were relatively small, but the study suggested that everolimus/tacrolimus-based immunosuppression was safe and effective and gave good renal function at 6 months.
In a global study performed in 13 countries (ASSET) everolimus was given along with tacrolimus (target levels of 4–7 ng/mL) for the first 3 months in all patients and thereafter the two study arms comprised one maintained at the same target level of tacrolimus and a tacrolimus minimization group with target tacrolimus levels of 1.5 to 3.0 ng/mL. The study failed to demonstrate any benefit in the tacrolimus minimization group, possibly as a result of overlapping of achieved tacrolimus exposure in the two groups, but suggested that everolimus in combination with early tacrolimus minimization was associated with a low rejection rate, good graft survival and renal function, and an acceptable safety profile.
The lower rejection rate in ASSET is in contrast to the findings of a more recent noninferiority study, where everolimus (target 3–8 ng/mL) and low-dose tacrolimus (4–7 ng/mL) were compared with MMF and standard dose tacrolimus (8–12 ng/mL) in 613 recipients of deceased donor kidney transplants, including extended criteria and circulatory death donor kidneys. This study showed inferiority in the composite endpoint (biopsy-proven rejection, graft loss, and death) of the everolimus/tacrolimus arm; The incidence of biopsy-proven acute rejection was greater (19.1% EVL + LTac, 11.2% Tac + MMF, P < 0.05) although graft loss (1.3% EVL + LTac, 3.9% Tac + MMF, P < 0.05) was less with the everolimus plus low-dose tacrolimus combination. There was no difference in MDRD GFR at 1 year.
In conclusion, there is no convincing evidence that use of mTOR inhibitors as de novo therapy offers any advantage over CNI-based therapy. The 2009 KDIGO clinical practice guidelines for the care of kidney transplant recipients recommend that the combined use of mTOR inhibitors and CNIs should be avoided because they potentiate nephrotoxicity, particularly if used in the early period after transplantation.
Maintenance Therapy with mTOR Inhibitors
Although available data suggest that mTOR inhibitors are almost as efficacious in terms of immunosuppressive potency as CNIs when used as the principal immunosuppressive agent, their use immediately after renal transplantation is undesirable because of their effects on wound healing and lymphocele formation. Because mTOR inhibitors used in the absence of CNIs do not have the same nephrotoxicity concerns as CNIs they are potentially attractive agents for use in the maintenance phase of the posttransplant course, especially in patients with CNI-associated problems, including chronic allograft nephropathy. The first major study to examine the efficacy of mTOR inhibitors in this context used sirolimus combined with cyclosporine and steroids as initial therapy, with the cyclosporine being stopped at 3 months in half the patients. Sirolimus was shown to provide sufficient immunosuppression during the maintenance phase, with superior calculated creatinine clearance compared with patients remaining on sirolimus and cyclosporine. Although the acute rejection rate was slightly higher in the no-cyclosporine group, this did not translate into poorer renal function. Longer follow-up confirmed the sustained benefit of sirolimus maintenance therapy. This study had no standard control group, and the subsequent finding of enhanced nephrotoxicity when CNIs are combined with sirolimus casts a shadow over the results.
Smaller studies also suggested a benefit of sirolimus over CNIs as maintenance therapy after renal transplantation. A dual-center randomized controlled trial suggested that conversion to sirolimus in patients with impaired graft function results in a rapid improvement in measured glomerular filtration rate at 3 months, which was sustained to 2 years, whereas patients who remained on CNIs experienced deteriorating graft function. Because of concerns about triggering acute rejection during the conversion from CNIs to sirolimus, some investigators have used a period of overlap of immunosuppression, or covered the transition period with additional agents such as basiliximab, but in patients who are greater than 6 months posttransplantation, it is unlikely that this is necessary.
Late conversion to sirolimus is associated with three dominant side effects that might limit its usefulness as a maintenance agent, in addition to the other side effects that are well recognized with sirolimus (see later). First, more than half of patients in some studies experience a rash, either an acneiform rash or a dermatitis-like rash affecting the hands and, in particular, the fingers. Second, the period of conversion to sirolimus is associated with the development of mouth ulcers that, in most patients, resolve within 4 weeks. If such ulcers fail to resolve, herpes simplex should be considered. Finally, patients with suboptimal renal function, particularly patients with proteinuria, are prone to develop marked proteinuria after conversion (see later).
Despite the potential drawbacks in terms of side effects, evidence is accumulating that conversion from CNIs to mTOR inhibitors may be worthwhile in patients with interstitial fibrosis and tubular atrophy or histologic signs of CNI toxicity and, at least in the short term, may lead to improved graft function. The optimal time for conversion in such patients is unclear, but early rather than late conversion may be best, before irreversible fibrosis and tubular atrophy become extensive. With the increasing use of organs from more marginal donors that already have some damage at baseline, cessation of CNIs and replacement with mTOR inhibitors may become a useful strategy to prevent further decline in graft function from a suboptimal baseline. Switching to mTOR inhibitors in patients who are experiencing other side effects from CNIs, such as neurotoxicity and diabetes, may also be a reasonable option. Conversion from CNIs to mTOR inhibitors for patients who develop hemolytic-uremic syndrome may also be considered, although sirolimus itself has been identified as a cause of this condition. Because mTOR inhibitors lead to an increased urinary excretion of uric acid, a further possible indication for use of mTOR inhibitors in the management of severe gout in patients taking CNIs.
The 3C study was a two-step trial, looking first to compare the incidence of rejection in patients receiving either alemtuzumab or basiliximab induction, followed by a second randomization at 6 months to either sirolimus-based immunosuppression or continuation on tacrolimus. The alemtuzumab arm had no steroids and lower dose tacrolimus, in addition to mycophenolate sodium (MPS); the basiliximab arm had full dose tacrolimus, MPS, and steroids. The study enrolled 852 patients, and the initial phase of the study showed that alemtuzumab was associated with less rejection but no difference in transplant survival at 6 months. In the subsequent randomization, 197 patients were allocated to stay on tacrolimus whereas 197 switched to sirolimus. There was no difference in GFR at 18 months (12 months postrandomization) but a significantly higher (RR 5.15) incidence of acute rejection in the sirolimus-treated patients (14.7% SRL, 3% Tac, P < 0.001); the incidence of rejection on sirolimus was no different whether the patient had alemtuzumab or basiliximab induction. In addition, 49% of patients discontinued sirolimus-based therapy compared with 6% tacrolimus-based therapy after randomization. Serious infections were also more common on sirolimus (48.2% SRL, 35.5% Tac, P = 0.008). The high discontinuation rate on sirolimus and the high rejection rate probably account for why the patients randomized to sirolimus saw no benefit in GFR on an intention-to-treat analysis.
The ZEUS study, a multicenter, open-label, randomized controlled trial of everolimus-based, CNI-free immunosuppression suggested that early replacement of CNIs with everolimus-based immunosuppression is advantageous for renal function. The study recruited 503 adult recipients of renal transplants, and they received initial therapy with basiliximab induction together with cyclosporine, mycophenolate, and prednisolone. At 4.5 months after transplantation recipients were randomized ( n = 300 or 60% of the total recruited) to continue on cyclosporine ( n = 145) or switch to an everolimus-based regimen ( n = 155) with everolimus trough concentrations of 6 to 10 ng/mL. Those recipients who were switched to everolimus showed a significant improvement in 12-month GFR (the primary endpoint) compared with those who remained on cyclosporine (71.8 mL/min/1.73 m 2 vs. 61.9 mL/min/1.73 m 2 ). Conversion to everolimus was associated with a 6% higher rate of postrandomization acute rejection than remaining on cyclosporine (10% vs. 3%) but over the entire study period acute rejection rates were similar (15%). The overall efficacy and safety were similar in the two study groups. Five-year follow-up of the ZEUS study showed a sustained benefit in terms of renal function in the everolimus arm (intention-to-treat: Nankivell GFR 66.2 mL/min/1.73 m 2 in EVL arm, 60.9 mL/min/1.73 m 2 in CsA arm), with greater benefit when analyzed in terms of those remaining on everolimus. This benefit is in spite of the cumulative incidence of acute rejection postrandomization over the 5-year follow-up period being nonsignificantly higher in the everolimus arm (13.6% EVL, 7.5% CsA P = 0.095).
The ZEUS trial contrasts with the results of ELEVATE, a similar large multicenter trial randomizing 715 de novo patients to stay on their CNI or switch to everolimus at 3 months. Patients received basiliximab induction and either tacrolimus or cyclosporine-based immunosuppression together with MPS and steroids: 360 were randomized to everolimus, with 357 randomized to CNIs, of which 231 were on tacrolimus and 125 on cyclosporine, with one dropout starting on renal replacement in each arm. The primary endpoint was change in MDRD GFR at 12 months, and there was no significant difference between study arms (0.3 mL/min/1.73 m 2 EVL, −1.5 mL/min/1.73 m 2 CNI). However, there was a significantly better estimated glomerular filtration rate (eGFR) at 12 and 24 months in everolimus-treated patients (24-month GFRs: 62.5 mL/min/1.73 m 2 EVL, 57.4 mL/min/1.73 m 2 CNI, P = 0.005). There was a greater proportion of biopsy-proven acute rejection on everolimus compared with CNI overall (9.7% EVL, 4.8% CNI, P = 0.014); when the CNIs were analyzed separately, everolimus- and cyclosporine-treated patients had a similar incidence (8.8%), whereas tacrolimus patients had a significantly lower incidence (2.6%) than everolimus-treated patients. As with other mTOR studies, there were more discontinuations in the everolimus group than the CNI group (23.6% EVL, 8.4% CNI). Other observations were a significantly greater mean urinary protein/creatinine ratio in the everolimus patients than the CNI patients (36.4 mg/mmol EVL, 19.1 mg/mmol CNI, P < 0.001), with no difference between tacrolimus- and cyclosporine-treated patients.
mTOR Inhibitors and Malignancy
mTOR Inhibitors as Antitumor Agents
The P13K/Akt signaling pathway is often dysregulated in malignant cells, causing activation of mTOR and thus stimulating cell proliferation, tumor growth, and production of growth factors, such as vascular endothelial growth factor, that stimulate angiogenesis, a key component of many tumors. Because of the central role of mTOR in regulating cellular processes in malignant cells it is an attractive therapeutic target. Although intuitively the immunosuppressive effects of mTOR might be expected to outweigh any beneficial effect on limiting tumor cell growth in patients with malignancy, this seems not to be the case and a number of new sirolimus derivatives (rapalogs) have been developed specifically for use as antitumor agents, including temsirolimus (CCI-779), a sirolimus derivative formulated for intravenous administration. mTOR inhibitors have been licensed for a number of different tumors, including renal cell carcinoma, gliomas, neuroendocrine tumors, and advanced hormone receptor positive/HER2 negative breast cancer. The use of mTOR inhibitors is currently being tested in many of the other common types of cancer including colorectal, ovarian, prostatic, gastric, and pancreatic cancer.
mTOR Inhibitors and Posttransplant Malignancy
Because patients after renal transplantation are at increased risk of developing most types of malignancy, particularly squamous cell cancer of the skin and lymphoma, the anticancer effects of mTOR inhibitors are of great relevance. Several reports suggest that maintenance immunosuppression with mTOR inhibitors after renal transplantation may be associated with a reduced risk of posttransplant malignancy. It is, however, important to bear in mind that this could, at least in part, reflect the ability of CNIs to increase the risk of malignancy rather than any direct protective effect of mTOR inhibitors. A multivariate analysis of posttransplant malignancy in 33,249 renal allograft recipients in the US revealed that the incidence rates of any type of posttransplant malignancy were 0.6% in patients taking mTOR inhibitors, 0.6% for patients taking mTOR inhibitors plus CNIs, and 1.8% for patients taking CNIs alone. Similarly, the incidence of posttransplant malignancy in adults randomly assigned to remain on sirolimus and CNIs was found to be greater than in subjects randomly assigned to early CNI withdrawal and an increased dose of sirolimus.
A more recent meta-analysis of 20 randomized controlled trials and two observational studies, comprising 39,039 patients, suggested there was a protective effect of sirolimus, at least for nonmelanoma skin cancer (incidence rate ratio [IRR] 0.49; 95% CI 0.32, 0.76), particularly where the comparator was cyclosporine. There was also a suggestion of a lower incidence of kidney cancer (IRR 0.40; 95% CI 0.20, 0.81) and a higher rate of prostate cancer (IRR 1.85; 95% CI 1.17, 2.91). In a separate, earlier analysis of 21 trials and 5876 patients, a similar effect was seen for sirolimus in reducing the incidence of nonmelanoma skin cancer (NMSC), particularly where the patient was converted to sirolimus from another regimen. This study also showed a reduction in the risk of other cancers (risk reduction [RR] 0.52, 0.38–0.69), but worryingly indicated an increased risk of death on sirolimus compared with controls.
mTOR Inhibitors and Nonmelanoma Skin Cancer
The most frequent type of cancer after renal transplantation is NMSC, which affects over half of all transplant recipients and is an important consideration when considering the optimal immunosuppressive regimen, especially for patients considered at particularly high risk because of previous skin cancers or premalignant skin lesions. One of the earliest reports in this context was a single-center randomized controlled trial that showed that renal transplant recipients with premalignant skin lesions who were switched to sirolimus-based immunosuppression had less progression and in some cases even regression of premalignant skin lesions. There were 25 patients assigned to the sirolimus arm and 19 to the control arm, and of the 9 patients who developed histologically confirmed NMSC during the 12-month follow-up period, only one was in the sirolimus arm. Another multicenter study (the TUMORAPA study) randomly assigned renal transplant recipients who had at least one invasive posttransplant cutaneous squamous cell carcinoma either to remain on CNI-based immunosuppression or to switch to sirolimus. New squamous cell cancers developed in 14 (22%) of those who switched to sirolimus and 22 (39%) of those randomized to stay on calcineurin blockers with a median time to onset of 16 versus 7 months respectively ( P = 0.02). Switching to sirolimus appears to be an effective strategy for reducing the risk of recurrent skin cancer and is a reasonable treatment option although not without potential side effects. In the TUMORAPA study, twice as many serious adverse events occurred in those who switched to sirolimus and 23% of patients discontinued sirolimus because of adverse events.
mTOR Inhibitors and Posttransplantation Lymphoproliferative Disorder
Everolimus and sirolimus have been shown to markedly inhibit the growth of human posttransplant lymphoproliferative disorder (PTLD)-derived cell lines and Epstein–Barr virus-transformed B lymphocytes in vitro and in vivo. Nevertheless there is relatively limited evidence to support the use of mTOR inhibitors in the management of PTLD, although a renal transplant recipient in whom disseminated PTLD resolved completely after conversion of immunosuppression to sirolimus has been reported. In a pooled analysis from nine European centers, 19 renal transplant recipients with PTLD were studied after conversion to mTOR inhibitors and subsequent withdrawal or minimization of CNIs. Remission of disease was observed in 15 of the recipients, although because 12 of the recipients also received rituximab or chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) it is difficult to know how much of the benefit was directly attributable to use of mTOR inhibitors. Sirolimus did not appear to modify the risk of developing PTLD in an analysis of 25,127 patients who underwent renal transplantation in the US, 34 of whom developed PTLD.
mTOR Inhibitors and Kaposi’s Sarcoma
mTOR inhibitors may have a useful role in the treatment of renal transplant recipients who develop Kaposi’s sarcoma associated with herpesvirus-8, especially if the disease is confined to the skin. In a study of 15 patients who developed cutaneous Kaposi’s sarcoma after renal transplantation while taking cyclosporine, switching them to sirolimus led to complete, histologically confirmed remission in all patients for the duration of the study (6 months) with preservation of graft function. However, response varies and may depend on the severity of disease. A retrospective analysis in which 14 renal transplant recipients with Kaposi’s sarcoma (including several with visceral or advanced disease) were switched from CNIs to sirolimus showed that the switch was generally well tolerated. Complete remission was seen in two patients, and a partial response was seen in a further eight, although three of the partial responders with advanced disease relapsed after several months. In patients who do not respond to mTOR inhibitors or show significant side effects it has been suggested that a combination of mTOR inhibitor and leflunomide (which inhibits Akt signaling upstream from mTOR) may act synergistically in the treatment of Kaposi’s sarcoma. The use of mTOR-based immunosuppression, however, does not necessarily prevent the development of posttransplant Kaposi’s sarcoma. Further studies are needed to evaluate the role of mTOR inhibitors in the treatment of Kaposi’s sarcoma and to determine the optimal treatment strategy for patients with more advanced disease.
mTOR Inhibitors and BK Virus
Polyoma virus nephropathy caused by BK virus is a well-recognized cause of kidney graft dysfunction. The incidence in patients with sirolimus-based immunosuppression has been reported to be relatively low, compared with other immunosuppressive regimens, although this reduced incidence may not be the case where mTORs are combined with CNIs. Early reports that a sirolimus-based regimen may be associated with early resolution of nephropathy have been confirmed by later reports using everolimus-based regimens.
These observations are supported by in vitro evidence suggesting that sirolimus may be working by blocking intracellular protein kinase pathways used by the virus, an effect that is complemented by the addition of leflunomide. Clinical efficacy of the combination of everolimus and leflunomide has been reported in two cases.
mTOR Inhibitors in Polycystic Kidney Disease
Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder characterized by the development of cysts in the kidneys that results in loss of functioning nephron mass such that patients develop kidney failure in adulthood. Patients with ADPKD frequently have cysts in other organs, such as the liver that can compromise hepatic function. Studies have implicated mTOR in cyst pathogenesis, and mTOR inhibitors have been shown to slow cyst development in animal models. In human patients with ADPKD who went on to receive a kidney transplant, mTOR inhibition was associated with a reduction in hepatic cysts in some subjects. Randomized controlled trials of mTOR inhibitors in nontransplanted ADPKD patients have failed to demonstrate efficacy in preventing cyst formation. However, recent data suggest that combined phosphoinositide 3-kinase (PI3K) and mTOR inhibition may be a more effective strategy, but this remains to be tested in patients.
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