Sirolimus therapy in cardiac transplantation

Sirolimus Therapy in Cardiac Transplantation B. Radovancevic and B. Vrtovec ABSTRACT Rapamycin powerfully inhibits the progression of antigen-activated T cells through the cell cycle. In animal heart transplantation models, rapamycin therapy has been associated with profound immunosuppressive effects on host humoral and cellular responses. In consequence, further studies have been conducted to evaluate the efficiency of rapamycin in preventing acute heart allograft rejection, treating refractory acute heart allograft rejection, inducing transplantation tolerance, and preventing and treating transplant coronary artery disease. The results of these studies indicated that rapamycin can effectively prevent acute graft rejection and inhibit refractory acute graft rejection in heart transplant recipients by exerting potent immunosuppressive and antiproliferative effects without adversely affecting renal function. This supports the use of rapamycin therapy in heart transplant recipients, especially in those with renal dysfunction, for whom treatment with calcineurin inhibitors is contraindicated. Rapamycin may also halt and even reverse the progression of cardiac allograft vasculopathy, which warrants further clinical trials in humans. Finally, rapamycin may be able to induce transplantation tolerance, thus making it one of the most promising modalities for improving the long-term survival of heart transplant recipients.

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IROLIMUS (rapamycin) is a macrocyclic triene antibiotic produced by the actinomycete Streptomyces hygroscopicus.1 Although originally used as an antifungal agent, it is now gaining wider acceptance as a novel immunosuppressive agent in solid organ transplantation. Via its C-7 methoxy group, rapamycin crosslinks the immunophilin FK binding protein 12 (FKBP12) to a serine-threonine kinase termed mammalian target of rapamycin (mTOR).2 The resulting blockade of mTOR alters various signal transduction pathways leading from cytokine or growth-factor stimulation to the G1-to-S phase transition, which ultimately blocks progression through the cell cycle in the late G1-to-S phase.3 In this way, rapamycin inhibits the proliferation of lymphoid cells (T and B lymphocytes) and nonlymphoid cells (endothelial cells, smooth muscle cells, fibroblasts) and enhances apoptosis in various types of tissues.4 Rapamycin powerfully inhibits the progression of antigen-activated T cells through the cell cycle. Furthermore, it has been shown to selectively inhibit production of cytotoxic but not noncytotoxic antibodies and to preferentially activate T helper 2 cells in a rat heart transplantation model.5 In presensitized rat cardiac transplant recipients, rapamycin therapy has been associated with profound immunosuppressive effects on host humoral and cellular responses.6 In both rat7 and rabbit8 models, it has been efficient in © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35 (Suppl 3A), 171S–176S (2003)

preventing acute cardiac allograft rejection, presumably because of its combined cellular and humoral immunosuppressive properties. Like cyclosporine and tacrolimus, rapamycin initially binds to an intracellular protein of the immunophilin family.9 However, the resulting complexes have different targets. When bound, respectively, to cyclophilin and FKBP12, cyclosporine and tacrolimus interact with and inhibit the activity of calcineurin, a calcium-regulated phosphatase. In contrast, when rapamycin binds to FKBP12, the resulting complex does not affect calcineurin activity, which suggests a good potential for inducing complementary immunosuppressive effects via calcineurin inhibition and rapamycin treatment. Indeed, calcineurin inhibitors and rapamycin have been shown to exert synergistic beneficial effects on cardiac allograft survival in a rat model.10 These synergistic effects appear to be due to the intracellular abundance of FKBP12, a condition that greatly lessens the competition between the calcineurin inhibitors and rapamycin for binding. From the Texas Heart Institute at St. Luke’s Episcopal Hospital, Houston, Texas. Address reprint requests to B. Radovancevic, Texas Heart Institute, P.O. Box 20345, MC 2-114, Houston, TX 77225-0345. 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00229-X 171S

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Fig 1. Rejection and infection rates in heart transplant recipients treated with rapamycin (rapamycin group) versus standard triple immunosuppressive therapy (control group).

Encouraging preliminary studies in animal models have demonstrated the important immunosuppressive role of rapamycin both alone and as an adjunct to calcineurin inhibitor treatment. In consequence, further studies have been conducted to evaluate the efficiency of rapamycin therapy in (1) preventing acute heart allograft rejection; (2) treating refractory acute heart allograft rejection; (3) inducing transplantation tolerance; and (4) preventing and treating transplant coronary artery disease. PREVENTION OF ACUTE HEART ALLOGRAFT REJECTION

The efficiency of de novo rapamycin therapy in preventing acute allograft rejection in human heart transplant recipients was first tested in a pilot clinical trial in 2001.11 Eleven heart transplant recipients received an initial rapamycin loading dose of 10 to 15 mg followed by a daily dose of 10 mg up to a target plasma level of 10 ng/mL. All patients were treated with a standard prednisone regimen, and eight also received basilixicimab (20 mg) on postoperative days 1 and 4. On average, cyclosporine (Neoral) therapy was initiated on postoperative day 13. The control group included 142 heart transplant recipients who were treated with a standard triple-drug immunosuppression therapy consisting of cyclosporine, prednisone, and either azathioprine or mycophenolate mofetil. Compared with controls, rapamycin-treated patients had a lower rejection rate (8% vs 52%) and a lower mean number of rejection episodes per

individual (0.2 vs 0.9). No intergroup difference in infection incidence was noted. According to this pilot clinical trial, rapamycin effectively prevented acute allograft rejection in heart transplant recipients without concomitantly increasing posttransplantation infection rates (Fig 1). Moreover, a delay in initiation of cyclosporine therapy and a decrease in the maintenance dosage of cyclosporine in this study (Fig 2) resulted in significantly improved renal function, thus further emphasizing the benefits of using rapamycin in heart transplant recipients early after transplantation. Similarly, rapamycin was shown to be a safe and effective treatment modality in tacrolimus-treated heart transplant recipients.12 Patients treated with sirolimus, tacrolimus, and steroids had fewer rejection episodes, lower doses of tacrolimus, lower creatinine levels, and higher triglyceride levels than did heart transplant recipients treated with mycophenolate mofetil, tacrolimus, and steroid therapy. In light of these encouraging results, a randomized, controlled trial of rapamycin immunosuppressive therapy in a larger cohort of patients is warranted. TREATMENT OF REFRACTORY ACUTE ALLOGRAFT REJECTION

Rapamycin therapy has been proposed by some as a useful adjunct in the treatment of acute allograft rejection that is not reversible with a standard treatment regimen. The rationale for this is that (a) rapamycin in vitro displays an immunosuppressive potency 10 to 100 times greater than

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Fig 2. Mean posttransplantation levels of cyclosporine in heart transplant recipients treated with rapamycin (rapamycin group) versus standard triple immunosuppressive therapy (control group).

that of calcineurin inhibitors13; (b) rapamycin and calcineurin inhibitors have different sites of action2; and (c) rapamycin appears to be less nephrotoxic than calcineurin inhibitors.11 In animal models, rapamycin has been highly successful in reversing the ongoing refractory acute rejection of heart allografts.5,14 Consequently, rapamycin as a rescue therapy in patients suffering acute heart allograft rejection has been tested in both pediatric and adult heart transplant recipients. Pediatric transplant recipients treated with calcineurin inhibitors experience a higher incidence of posttransplantation lymphoproliferative disorder and end-stage renal dysfunction.15 However, two reports suggest that rapamycin-based immunosuppression may be an attractive alternative to standard immunosuppressive modalities in this population. In one, rapamycin (mean dose 2 mg/d) successfully reversed refractory allograft rejection without causing significant adverse effects in five pediatric heart transplant recipients originally treated with tacrolimus.16 Similarly, in a case report of a pediatric heart transplant recipient with severe acute allograft rejection that did not abate despite treatment with antithymocyte serum, corticosteroid pulses, methotrexate, prednisone, mycophenolate mofetil, and tacrolimus, rapamycin therapy rapidly resolved the cardiac rejection.17 Yet despite this promising preliminary evidence, larger studies are needed to investigate further whether rapamycin can be used safely and efficiently to

treat ongoing acute refractory rejection in the pediatric heart transplant recipient population. Rapamycin also holds promise for adult heart transplant recipients. One report has described the use of rapamycin as rescue therapy in a heterogeneous group of 13 patients whose traditional posttransplantation therapies were deemed very likely to lead quickly to graft failure, permanent dialysis, severe neurotoxicity, or death.18 A subset of these patients (n ⫽ 6) was prescribed rapamycin for refractory acute rejection and experienced no further documented episodes of rejection. However, the same subset also had an increased incidence of infection (37 infectious complications in 21 patients), thus emphasizing the importance of adequate rapamycin dosing, particularly in the setting of a multiple immunosuppressive-drug regimen. Together, these findings indicate that rapamycin can be a powerful immunosuppressant in adult heart transplant recipients in whom standard therapy has failed or is inappropriate.19 To achieve a favorable balance between rejection suppression and infection incidence in this setting, however, measurement of serum rapamycin levels may be beneficial.4 INDUCTION OF TRANSPLANTATION TOLERANCE

Although advances in immunosuppressive therapy have led to improved posttransplantation survival, the central longterm aim of posttransplantation therapy remains transplantation tolerance. The most critical mechanism of transplan-

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tation tolerance is depletion of antigen-activated T cells.20 Therefore, drugs that interfere with the death of activated T cells (eg, cyclosporine) can inhibit tolerance, whereas drugs that induce their death can promote it. When not dividing, T cells become unresponsive when antigen recognition occurs without costimulation through the CD28 and CD154 accessory receptors. Thus, inhibitors of CD28 and CD154, together with inhibitors of T-cell division, represent the key elements in the induction of transplantation tolerance. Because of its antiproliferative effect on T lymphocytes, rapamycin has been tested as a potential tolerance-inducing agent in various animal models of heart transplantation. In hamster-to-rat21 and pig-to-rabbit22 xenograft models, rapamycin successfully prolonged cardiac graft survival when given in combination with other immunosuppressive agents. In mouse studies, rapamycin exerted, in addition to its inhibitory effect on T-cell division, tolerance-inducing effects that seemed to correlate with its capacity to block CD154 induction; this suggests that rapamycin may affect both early and late events in the process of T-cell activation.23 Together, these data support the potential use of rapamycin as a transplantation tolerance-inducing modality. PREVENTION AND TREATMENT OF CARDIAC ALLOGRAFT VASCULOPATHY

Because mTOR is widely distributed among signaling pathways in many tissues, the antiproliferative effects of rapamycin are not limited to lymphoid cells. Blockade of mTOR by rapamycin results in inhibition of cell cycle in several tissue types, including endothelial, fat, myocardial, skeletal muscle, macrophage, fibroblast, pancreatic, hepatic, bone marrow, and kidney cells.24 Because the growth and migration of vascular smooth muscle cells are important for neointimal proliferation after vascular injury, many studies have addressed the potential antiproliferative effects of rapamycin in vascular smooth muscle.25 In addition to its effects in modulating growth of vascular smooth muscle through cell-cycle inhibition, rapamycin has been shown to inhibit the migration of vascular smooth muscle cells without affecting their ability to bind collagen and without disrupting their cytoskeletal components.26 Furthermore, rapamycin has been shown to inhibit intimal hyperplasia after balloon angioplasty of the carotid27 and coronary28 arteries. These findings were the basis for studies of rapamycin as an agent for reducing in-stent restenosis in patients with coronary artery disease. In those studies, rapamycin-coated stents were extremely effective: patients who received rapamycin-coated stents had no angiographically proven restenosis at the 6-month followup.29 These data suggest that rapamycin may significantly attenuate the progression of atherosclerosis in patients with coronary artery disease. Cardiac allograft vasculopathy is generally characterized by the diffuse, concentric intimal thickening of both epicardial and intramural arteries.30 However, the observed spectrum of abnormalities is much broader, ranging from con-

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centric fibrous intimal thickening to diffuse, complex atherosclerotic plaques closely resembling native atherosclerosis.31 Studies in animal models have demonstrated that transplant vasculopathy can be markedly reduced with maintenance rapamycin therapy.32 Studies in small animal33 and nonhuman primate34 models have revealed an association between rapamycin treatment and the partial regression of cardiac allograft vasculopathy (Fig 3). These effects have been attributed to the antiproliferative effect of rapamycin on vascular smooth cells35 and have been shown to correlate closely with serum rapamycin levels.36 In addition, rapamycin may also have indirect effects on vascular smooth muscle cells. One of these may be its ability to upregulate nitric oxide synthetase, which results in higher local production of nitric oxide, a potent smooth muscle cell growth inhibitor.37 A second indirect effect may be the ability of rapamycin to reduce the formation of anti-HLA antibodies, which have been associated with classic cardiac allograft vasculopathy.33 Rapamycin’s ability to inhibit vasculopathy may also be explained in part by its effect on the kidneys. Cardiac allograft vasculopathy has been shown to correlate with hyperhomocysteinemia, whose primary cause seems to be renal failure.38 In addition, renal dysfunction is a common problem in heart transplant recipients, even in the absence of calcineurin therapy, because in most cases there is a long-standing pretransplantation history of congestive heart failure.39 Calcineurin inhibitors are nephrotoxic and may thus contribute to renal failure. In comparison, rapamycin appears to be much less nephrotoxic. This suggests that the use of rapamycin may allow calcineurin inhibitor therapy to be scaled back or even discontinued, which could lead to improved renal function. For these reasons, rapamycin may be preferable to calcineurin inhibitors in heart transplant recipients who show signs of renal failure or dysfunction. Despite its obvious benefit in inhibiting cardiac allograft vasculopathy, rapamycin is also associated with adverse effects. Rapamycin appears to interfere with lipid clearance from the blood, possibly by inhibiting hepatic uptake, lipoprotein lipase activity, and signal transduction in peripheral tissues by insulin or insulin-like growth factors.23 Two of the most common adverse side effects of rapamycin are hypercholesterolemia and hypertriglyceridemia, which occur in about 40% of transplant recipients.40 However, even though hyperlipidemia has been associated with an increased incidence of cardiac allograft vasculopathy, its presence does not appear to affect the ability of rapamycin to inhibit vasculopathy.41 This suggests that the combination of rapamycin with an adequate lipid-lowering drug regimen may optimize the vasculopathy-inhibiting ability of rapamycin. CONCLUSIONS

Rapamycin can effectively prevent acute graft rejection and inhibit refractory acute graft rejection in heart transplant recipients by exerting potent immunosuppressive and anti-

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Fig 3. (A) Mean intimal areas of aortic allografts from untreated and sirolimus-treated monkeys assessed by serial intravascular ultrasound (IVUS) studies at 3-week intervals. (B) Cross-sectional IVUS image from an untreated monkey showing concentric thickening of the intima (white lines) in the middle part of an aortic allograft on day 105. (C) Cross-sectional IVUS image from a middle segment of an aortic allograft from a sirolimus-treated monkey showing no intimal thickening on day 105. (From Ikonen TS, Gummert JF, Hayase M, et al. Transplantation 2000;70:971. Reproduced with permission.)

proliferative effects without adversely affecting renal function. This supports the use of rapamycin therapy in heart transplant recipients. especially in those with renal dysfunction, for whom treatment with calcineurin inhibitors is contraindicated. Rapamycin may also halt and even reverse the progression of cardiac allograft vasculopathy, which warrants further clinical trials in humans. Finally, rapamycin may be able to induce transplantation tolerance, thus making it one of the most promising modalities for improving the long-term survival of transplanted hearts. REFERENCES 1. Napoli KL, Taylor PJ: Ther Drug Monit 23:559, 2001 2. Kahan BD, Camardo JS: Transplantation 23:1181, 2001 3. Kahan BD: Expert Opin Pharmacother 2:1903, 2001 4. MacDonald A: Transplantation 72(suppl 12):S105, 2001 5. Ferraresso M, Tian L, Ghobrial R, et al: J Immunol 153:3307, 1994 6. Schmidbauer G, Hancock WW, Wasowska B, et al: Transplant Proc 27:427, 1995 7. Stepkowski SM, Tian L, Napoli KL, et al: Clin Exp Immunol 108:63, 1997 8. Fryer J, Yatscoff RW, Pascoe EA: Transplantation 55:340, 1993 9. Baran DA, Galin ID, Gass AL: Curr Opin Cardiol 17:165, 2002 10. Vu MD, Qi S, Xu D, et al: Transplantation 64:1853, 1997 11. Radovancevic B, El-Sabrout R, Thomas C, et al: Transplant Proc 33:3221, 2001 12. Pham SM, Qi XS, Mallon SM, et al: Transplant Proc 34:1839, 2002 13. Vu MD, Qi S, Xu D, et al: Transplantation 66:1575, 1998 14. Chen H, Wu J, Xu D, et al: Transplantation 56:661, 1993

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