Introducing Everolimus (Certican) in Organ Transplantation: An Overview of Preclinical and Early Clinical Developments

Abstract

Everolimus (Certican) is a novel proliferation signal inhibitor with potent immunosuppressant effects that has been shown to prevent and reverse acute rejection in preclinical models of kidney, heart, and lung transplantation. These effects have been confirmed by clinical studies in which everolimus demonstrated comparable efficacy to azathioprine and mycophenolate mofetil in heart and renal transplantation, respectively, when combined with full-dose cyclosporine (CsA; Neoral). Experimental studies have also shown that everolimus inhibits smooth muscle cell proliferation and prevents neointimal thickening and transplant arteriosclerosis. Importantly, everolimus was shown to significantly reduce both the incidence and severity of cardiac allograft vasculopathy in recipients of heart transplants, which if confirmed in long-term studies, could provide a major advantage in these patients. Use of everolimus was also associated with lower rates of cytomegalovirus infection in both indications. Studies in which everolimus was combined with reduced-exposure CsA have demonstrated significantly improved renal function when compared with full-dose regimens, with no loss of efficacy. The results of preclinical and clinical studies with everolimus thus indicate that this novel proliferation signal inhibitor will be an important addition to the armamentarium by enhancing graft survival and minimizing toxicity.

Everolimus (Certican, Novartis Pharma AG, Basel, Switzerland) is an orally-active proliferation signal inhibitor derived from the naturally-occurring immunosuppressive macrolide, rapamycin (sirolimus). Interestingly, the mode of action of these agents differs from that of the calcineurin inhibitors (CNIs); CNIs inhibit T-cell proliferation by blocking transcriptional activation of early T-cell specific genes, therefore inhibiting the production of T-cell growth factors like interleukin-2 (IL-2). In contrast, everolimus acts on a later stage of the T-cell cycle, blocking the proliferative signal provided by such growth factors. It therefore arrests cell division at the late G1 stage, preventing them from entering the S phase (¹). In preclinical studies, a synergistic immunosuppressive effect has been demonstrated between everolimus and cyclosporine (CsA; Neoral), possibly due to their complementary modes of action (²). Furthermore, everolimus has been shown to prevent vascular remodeling (³), a key component of progressive allograft dysfunction that has been linked to repetitive cycles of cytokine release, upregulation of growth factors, and vascular smooth muscle proliferation (Fig. 1) (⁴). Everolimus may therefore have the ability to target the primary causes of rejection after organ transplantation. This article provides an overview of the pharmacodynamics and pharmacokinetic effects of everolimus, and reviews the results of initial clinical studies.

FIGURE 1.: Vascular remodeling in transplanted organs, a key component of allograft dysfunction. Continued immune and endothelial activation posttransplant leads to cytokine release and upregulation of growth factors.

Antirejection and Antiproliferative Effects in Preclinical Models

Everolimus has been shown to prevent and reverse acute rejection in preclinical models of kidney, heart, and lung transplantation, and to inhibit the manifestations of chronic rejection that may contribute to graft loss (^{1–3, 5–8}). As previously stated, everolimus also synergizes with CsA to produce efficacy greater than either drug alone. For example, in cardiac and renal transplantation models in vivo, the minimum effective doses required to prolong allograft survival for more than 100 days were greater than 5 mg/kg for everolimus and 5 mg/kg for CsA (²). However, combination of everolimus and CsA led to a reduction in the minimum effective doses of both agents (0.5 or 1.0 mg/kg for everolimus and 1.0 mg/kg for CsA).

The ability of everolimus to prevent the manifestation of chronic rejection has been demonstrated in a model of chronic renal allograft rejection in rats (⁷). In this model, proteinuria (an indicator of chronic rejection) and other signs of chronic rejection were significantly reduced following everolimus treatment. Chronic rejection is frequently characterized by vascular remodeling, resulting in thickening of the intima of graft blood vessels. Experimental studies have shown that everolimus inhibits smooth muscle cell proliferation and prevents neointimal thickening and transplant arteriosclerosis (^{6, 9, 10}). In particular, the development of chronic rejection, assessed by intimal thickening, was reduced by everolimus (0.31–1.25 or 2.5 mg/kg/day) in rat models of vascular remodeling (³) and arteriosclerosis (⁶). Oral everolimus (1.5 mg/kg given 1 day before stenting, followed by 0.75 mg/kg/day for 28 days) has also been shown to suppress in-stent neointimal growth in rabbit iliac arteries (¹⁰). Studies are currently underway to determine whether similar effects can be demonstrated in man. If so, these effects could provide a major advantage for heart transplant patients, since vascular remodeling and neointimal proliferation are both key components of cardiac allograft vasculopathy, the primary cause of late morbidity and mortality in heart transplantation. A recent randomized trial in patients with de novo coronary lesions showed significantly lower in-stent late lumen loss and in-segment diameter stenoses in patients treated with everolimus-eluting stents than in those treated with metallic stents (¹¹).

Pharmacokinetics of Everolimus

The pharmacokinetics of everolimus have been assessed in stable renal transplant recipients receiving CsA-based immunosuppression (^12–14). These studies have demonstrated that oral everolimus is rapidly absorbed, with peak blood concentrations reached around 2 hr after dosing (^{12, 13}). Peak blood concentrations (C_max) and area under the curve (AUC) increase proportionally after a single dose of everolimus, and at steady state over the dose range of 0.75–1.5 mg bid (¹⁴). Everolimus is metabolized by the cytochrome P450 isozyme CYP3A4 and the P-glycoprotein counter-transporter, and has an elimination half-life of 28±7 hr in renal transplant patients (¹⁵). Everolimus has a shorter half-life than sirolimus (28 vs. 60 hr) and reaches steady state levels more rapidly (4 vs. 6 days) (¹⁶). Sirolimus and everolimus can exacerbate CsA nephrotoxicity, probably because of pharmacokinetic interactions (¹⁷) resulting in increased production of transforming growth factor β1 (¹⁸). The synergistic relationship between everolimus and CsA, does, however, permit use of lower CsA doses without loss of efficacy.

Everolimus in Heart and Lung Transplantation

The efficacy and safety of everolimus (1.5 or 3.0 mg/day) in heart transplantation was compared with azathioprine (AZA; 1.0–3.0 mg/kg/day) in a 2-year Phase III, multicenter, randomized study in 634 recipients (18–68 years) of primary heart transplants receiving concomitant full-dose CsA for microemulsion (Neoral), corticosteroids, and statins (¹⁹). Rates of acute rejection (Grade 3A or above) were significantly lower for patients receiving everolimus 1.5 or 3.0 mg/day (30.6% and 21.3%, respectively) than for those receiving AZA (46%; P<0.001). Furthermore, everolimus-treated patients had a significantly lower incidence of cytomegalovirus (CMV) infection at 12 months than AZA-treated patients (7.7% and 7.6% vs. 21.5%; P< 0.001). Intravascular ultrasonography was used in the study to assess coronary artery intimal proliferation at baseline and at month 12. Interestingly, both average intimal thickness and the incidence of cardiac allograft vasculopathy were significantly lower in patients receiving everolimus than in those receiving AZA at both 12 (¹⁹) and 24 months (²⁰). As cardiac allograft vasculopathy is the main risk factor for mortality after the first year posttransplant and acute rejection and CMV infection play a key role in its development (^21–23), this pivotal study suggests that primary immunosuppression including everolimus could provide important benefits for heart transplant patients.

The efficacy of everolimus in reducing the rate of progression of chronic rejection has also been demonstrated in lung transplantation in a randomized study of 213 maintenance patients receiving everolimus 3.0 mg/day, or AZA 1.0–3.0 mg/kg/day, in combination with CsA and steroids (²⁴). In lung transplantation, chronic rejection is manifested as bronchiolitis obliterans syndrome (BOS). Compared with patients receiving AZA, everolimus-treated patients had significantly reduced incidences of ΔFEV₁ >15% (decline in the forced expiratory volume in 1 second >15% of the reference value from the study entry value; 27.7% vs. 15.8%; P<0.05) and ΔFEV₁ >15% with BOS (20.5% vs. 8.9%) at 12 months posttransplant.

Everolimus in Renal Transplantation in Combination with Full-Dose CNIs

The efficacy of everolimus in renal transplantation has been compared with mycophenolate mofetil (MMF) in two large, 36-month, randomized, double-blind studies conducted in 583 (Study 1; B251) (²⁵) and 588 (Study 2; B201) (²⁶) de novo renal transplant patients also receiving full-dose CsA and steroids. Everolimus (1.5 or 3.0 mg/day) was as effective as MMF 2.0 g/day, in preventing graft rejection in both trials (Fig. 2), with no significant differences between groups for the incidence of the primary efficacy endpoint (biopsy-proven acute rejection [BPAR], graft loss, death or loss to follow-up) at 36 months (Study 1: 33.7%, 34.0% and 31.1%, respectively, and Study 2: 33.0%, 38.9% and 37.2%) (^{25, 26}). In Study 1, everolimus 1.5 mg/day was associated with a significantly lower incidence of antibody-treated acute rejection at 36 months compared with MMF (10% vs. 18%; P= 0.014) (²⁵), whereas a significantly lower incidence of late-occurring BPAR, graft loss, death, or loss to follow-up versus MMF was reported for this dose of everolimus at the same time point in Study 2 (1.2% vs. 5.9%; P< 0.05) (²⁶). Everolimus was well tolerated at both doses in the two studies, with similar incidences of adverse events between groups. Importantly, in Study 2, everolimus 1.5 mg or 3.0 mg/day was associated with a significantly lower incidence of CMV infection compared with MMF (6% and 7% vs. 20%, respectively; P< 0.05) (²⁶). Twelve-month data from Study 1 showed that patients treated with everolimus 1.5 mg or 3.0 mg had higher median creatinine values than patients receiving MMF (1.7 mg/dL and 1.8 mg/dL vs. 1.5 mg/dL, respectively) (²⁵). Mean creatinine clearance was also lower in everolimus-treated patients than in those receiving MMF. The protocol of these studies was thus amended to allow CsA dose reduction in a subset of everolimus-treated patients (see below).

FIGURE 2.: Comparable incidence of primary efficacy failure (biopsy-proven acute rejection, graft loss, death or loss to follow-up) at 12 and 36 months in de novo renal transplantation in two pivotal trials with everolimus in combination with full-dose cyclosporine (CsA) vs. mycophenolate mofetil (MMF) plus full-dose CsA (^{25, 26}).

In Combination with Reduced-Exposure CNIs

Following protocol amendment, CsA dose could be reduced to achieve a trough level of 100 ng/ml or less. In Study 1, 43 patients had their CsA dose amended. In the following 6 months, 26.1% of those receiving everolimus 1.5 mg and 50.0% of those receiving everolimus 3.0 mg had improvements in creatinine clearance of at least 10% (²⁵). These findings indicate that everolimus plus reduced-exposure CNI can result in improved renal function, without increasing the risk of rejection. This is supported by the findings of a 3-year, Phase II study that compared the efficacy and tolerability of everolimus 3.0 mg/day, in combination with basiliximab, steroids, and either full-dose or reduced-exposure CsA in 111 de novo renal transplant recipients (²⁷). In this study, efficacy failure was found to be significantly higher in the full-dose CsA than in the reduced-exposure CsA group at 6, 12, and 36 months (35.8% vs. 17.2%, respectively, at 36 months; P= 0.032). Mean creatinine clearance was also significantly higher in the group receiving reduced-dose CsA at 12 months (53.5 vs. 60.9 ml/min; P= 0.007).

Therapeutic Drug Monitoring of Everolimus in Renal Transplantation

A clear association has been demonstrated between everolimus trough concentrations and both risk of acute rejection and certain adverse events (e.g., hypertriglyceridemia, leukocytopenia, and thrombocytopenia) (²⁸). This suggests that therapeutic drug monitoring (TDM) of everolimus may provide a more optimal dosing strategy than fixed dosing. A retrospective analysis of patients enrolled in the two pivotal trials with everolimus in renal transplantation has been undertaken to investigate whether TDM may benefit patients receiving everolimus (²⁹). Everolimus trough blood levels of 3 ng/ml or greater were found to be associated with a reduced incidence of BPAR and graft loss compared with lower trough concentrations; hypertriglyceridemia and hypercholesterolemia also tended to increase with higher trough concentrations (Table 1). These studies identified 3 ng/ml as the minimum effective trough blood level of everolimus, and suggest that TDM is likely to improve outcomes in everolimus-treated patients (^{28, 29}). A target range for everolimus trough blood levels of 3–8 ng/ml was identified as being efficacious while minimizing risk of adverse events. With this in mind, an immunoassay kit (Innofluor Certican, Seradyn Inc.) has been developed to improve the convenience of everolimus TDM. This fluorescence polarization immunoassay will be widely available and is compatible with standard TDx machines; it allows reliable measurement of everolimus blood levels over the range 2–40 ng/ml.

TABLE 1: Incidence of efficacy and safety events in patients receiving everolimus according to trough concentration and in those receiving MMF following 12-month treatment (²⁹)

Phase IV Clinical Trial Program

The overall vision of the everolimus Phase IV program is to optimize its use with CNIs (CsA and tacrolimus), reduce immunosuppression-related complications, and investigate prevention of late graft loss. In the renal transplant setting, the program will feature trials focusing on CsA reduction/withdrawal, in combination with tacrolimus and steroid minimization. In heart transplantation, there will be trials in which patients with renal impairment are switched from CNIs to everolimus, and trials assessing the efficacy of everolimus with reduced-exposure CsA.

CONCLUSIONS

Clinical experience with everolimus has demonstrated that, when used with corticosteroids and full-dose CsA, it has comparable efficacy to AZA and MMF for reducing rates of acute rejection in heart and renal transplantation, respectively. Furthermore, use of everolimus was also associated with lower rates of CMV infection. Importantly, in renal transplant patients, no loss of immunosuppressive efficacy was observed when everolimus was combined with reduced-exposure CsA, and the regimen resulted in significantly improved renal function. When considered with everolimus’ ability to inhibit vascular remodeling, these findings indicate that this novel proliferation signal inhibitor will be an important addition to the armamentarium by enhancing graft survival and minimizing toxicity.