Sirolimus: a potential option for the prevention of chronic allograft nephropathy

Sirolimus: A Potential Option for the Prevention of Chronic A|lograft Nephropathy Josep M. Campistol

ver the past few decades, the continued progress in basic science, clinical medicine, and imnmnopharmacology has engendered exciting improvements in the field of renal transplantation. Today this procedure offers longer patient survival, improved quality' of life, and reduced medical expenses compared with dialysis. Consequently, renal transplantation is the theralu of choice for patients with end-stage renal disease. After the introduction ofcyclosporine into clinical practice in the early 1980s, l-year graft smMval rates in renal transplantation improved from 60% to between 80% and 90%, and patient survival increased to more than 90%? However, the incidence of acute graft rejection in the first 6 months remained high; approximately 40% of transplant recipients had at least 1 episode of acute rejection, w-' In the 1990s, the introduction of the new inamunosuppressive agents tacrolimus and mycophenolate mofetil led to a decrease in the incidence of acute rejection? .4 Although current l-year rates of patient and graft survival are excellent (95% and 90%, respectively), longterm results remain limited, and the results have not significantly improved in the last several years. ~:~ Consequently, a progressive increase in the waiting list for renal transplantation has been detected in all countries. Two reasons could explain the lack of long-term improvement in renal transplantation-chronic allograft nephropathy (CAIN) and death with a functioning graft, as a result of cardiovascular diseasesJ 1,7 Both remain leading causes of the late loss of renal allografts, resuhing in an annual rate of loss of 3% to 5%. For these reasons, strategies that may improve long-term results of renal transplantation, especially the prevention of CAIN, have become a priority in renal transplantation.

O

From the Renal Transplant Unit. Hospital Clinic, Univ,~'i!r of BarceIona. h~slitut d'Invest(~aciolL~Biom~diquesAugtL~lPi i Sunrer (IDIBAPS), Barcelona. Spain. Address reprint reque.~tstoJosep M. Cam/~istol,MD, PItD, Renal Tramplant Unit, Hospital CIh~ic, Villarroel 170, 08036 Barcelona, Spain. © ")003 Elsevier hu'..'Ill rights resen'ed. 0955-470.W 03/1701-0003530. 00/0 doi:10.1053/tn'e.2003.4

Chronic Allograft Nephropathy CAN is the most prevalent cause of late renal graft loss and has traditionally been recognized as the result of repeated low-grade immune responses directed against allogeneic tissues. Recent evidence, however, indicates that nonimmunologic or alloantigen-independent factors contribute to its pathogenesis. ~:~ Nonimmunologic factors that may promote the development of CAN include donor age, ischemia-reperfusion injury, arterial hs]Jertension, lipid abnormalities, and calcineurin inhibitor-induced nephrotoxicityY,l° Although there is some dispute over the association between calcineurin inhibitorinduced nephrotoxicity and CAN development, nephrotoxicity induced by cyclosporine and tacrolimus is likely to be one of the most sigaaificant causes of CAN./L.v' Both acute (functional) and chronic (structural) nephrotoxicity may contribute to the development of CAN in renal transplant patients treated with calcineurin inhibitors, and nephrotoxicity is directly related to their mechanism of action. CAN is manifested clinically by a gradual decrease in renal function accompanied by hypertension and low-grade proteinurig, usually occurring months or years after renal transplantationY ~Characteristic histopathologic features of CAN include obliterative intimal fibrosis in the arteries of the graft (transplant arteriolopathy), widespread duplication of the glomerular basement membrane (chronic transplant glomerulopathy), tubular atroplay, and interstitial fibrosis? 4 Not uncommonly, however, only interstitial fibrosis and tubular atrophy are present in biopsies of allografts with CAIN. Determining the optimal treatment for established CAN remains a challenge. To date, no immunosuppressive regimen has been shown to be effective tbr the treatment or prevention of this condition in clinical renal transplantation. Increasing the dose of calcineurin inhibitors is unlikely to be beneficial because of the nephrotoxicity associated with these agents. Preliminary clinical studies suggest that the addition of mycophenolate mofetil, with or without reduction ofcyclosporine doses, might be an effective strategy/'or stabilizing or improving allograft func-

Tran@lantation Reviews. Vol 17, No I C]anuao,), ")003: pp 11-I9

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Josep M. Campistol

tion in patients with established CAN) .~ Moreover, experimental and preliminary human studies have suggested a prominent role for sirolimus (SRL, rapamycin) in the treatment and prevention of CAN.

Cardiovascular Death With a Functioning Graft Data have now emerged that highlight the importance of death with a functioning graft as a major category of late allograft loss. "i Approximately 35% of graft losses are because of the death of recipients with a functioning graft. ,~.6 In general, the main cause of death in the renal transplant population is cardiovascular disease, primarily ischemic heart disease. Among the factors contributing to an increased risk of ischemic heart disease and death fi'om cardiovascular causes, older age of recipient and donor, obesity, arterial hypertension, hyperlipidemia, diabetes mellitus, long-term on dialysis therapy, and smoking appear to be predominant, j7 The roles of hyperhomocysteinemia and endothelial dysfunction are currently being evaluated. It is evident that some immunosuppressive drugs could have a detrimental effect on the development of cardiovascular disease in renal transplantation, inducing diabetes, h)q)ertension, or Iwperlipidemia. The prevention of cardiovascular morbidity and mortality" seems essential for long-term graft and patient survival? 8

SRL SRL, a macrolide antibiotic, is a natural fermentation product of the soil actinomycete Streptomvces h.ygroscopicus discovered in Rapa Nui (Easter Island). Initially investigated for its anticandidal activiu,, SRL was shown to possess immunosuppressive activity in vitro, t'j Interest in the drug as an immunosuppressive agent was kindled by the discovery and administration of the structurally related macrolide tacrolimus in patients undergoing solid-organ transplantation. Although SRL and tacrolimus are structural analogs, they have a different mechanism of action and safety profile. SRL has different abilities that could make it the optimal drug for the treatment and prevention of chronic rejection. These abilities are summarized in the following 6 items: mechanism of action, antiproliferative action, nonnephrotoxicity of drug, SRL-based therapy, cyciosporine withdrawal, and conversion from calcineurin inhibitors to SRL.

Mechanism of Action SRL has a unique naechanism of action among inamunosuppressive drugs. It inhibits the costimulatory pathways necessary for cytokine s)lathesis and protein synthesis, as well as deoxya'ibonucleic acid transcriptional processes mediating cell-c3,clc progression after cytokine stimulation. 2~ On unimpeded entry into the cTtoplasm , SRL lbrms complexes with the same immunophilin as tacrolimus, an FK-binding protein (FKBP), to form an active inhibitor of cytosolic processes (Fig 1).21 In the costimulatory cascade necessary for the G0-GI progression, SRLFKBP complexes inhibit the activity ofc-rel, a crucial intermediate in the amplification of signal 1 activating the T-cell response."'-' After autocrine and paracrine reception of the cytokine signal, a muhifunctional kinase--manamalian target of rapamycin (mTOR)--exerts a variety of actions that regulate the phosphorylation of several sarcoma-like, receptor-type, and cell-cycle-dependent kinases. The SRL-FKBP-mTOR complex inhibits p70-$6 kinase, which leads to hylgerphoslghorylation of 40S ribosomal proteins. 2:~ Inhibition of roTOR disrupts the dissociation of the elongation initiation factor, which is necessarT for protein synthesis and for hyperphosphorylation of retinoblastoma protein, a crucial factor in cell-cycle progression. A fourth effect of SRL inhibition of roTOR is to prevent activation of downstream serine-threonine protein kinasesY ~ SRL blocks Ca++-dependent and Ca++-independent activation pathways mediating transduction of proliferative and differentiation signals delivered by the lymphokines interleukin (IL) 2, IL-3, IL-4, IL-5, IL-6, and IL-15 that act on T and B cells. '-'~.'-':~The net effect of the items mentioned previously results in inhibition ofT-cell proliferation. In B cells, SRL blocks not only cytokine-dependent but also Staplg,lococcus attreusstimulated and soluble CD4 ligand-stimulated proliferative signals. SRL exerts a unique action to interrupt immunoglobulin class switching. 25

Antiproliferative Actions of SRL There are several in vitro and in vivo experiences that showed the potent antiproliferative actMty of SRL. In 6 different models involving endothelium, aortic smooth muscle cells, cardiac fibroblast, and bovine smooth muscle cells, SRL has been sho~ql to inhibit cytokine-driven proliferation.2~-3~ SRL prevented nonstimulated vascular smooth muscle cells from Gf~ to Gi progression, apparently caused by

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inhibited retinoblastoma protein phosphorylation associated with a decrease in p33 c'Kk2.Fibroblast growth factor/3 stimulated in bovine or human endothelial cell cultures failed to display proliferation responses in the presence of SRL. 29 Moreover, SRL blocks exogenous stimulation of cardiac fibroblasts by different growth factors. ~ These in vitro models suggest that SRL may block the action of different cytokines critical to producing the immuno-obliterative vascular and bronchial lesions, which serve as histopathologic hallmarks of chronic rejection. SRL also has a beneficial effect on vascular injury responses in vivo (Fig 2). In 2 different models, cellular proliferation reactions have been shown to be dampened by the treatment with SRL, and Cooper et aP 2 and Gregory et ala3 noted that SRL treatment reduced the degree of intimal thickening in response to balloon catheter injury. This inhibitory effect was observed in stents coated with SRL for the prevention of restenosis after revascularization, s4 Morice et

aP 5 recently showed in a multicenter study of more than 200 patients with coronary stenosis that the SRL-eluting stent has considerable promise for the prevention of neointimal proliferation, restenosis, and associated clinical events, as compared with the standard coronary stent. 35 In the alloimmune model of rat aortic interposition grafts, Geerling et aP 6 observed a reduced adventitial inflammatory infiltration, presumably in response to cytokines, when treated with SRL. 36 On the basis of SRL's immunosuppressive effects on the rejection ofvascularized pig, rat, and nonvascularized mouse allografts, Meiser et al37 reported that SRL mitigates chronic rejection in a rat model. These preliminary findings were confirmed by Schmid et al, who determined that the extent of the immunosuppressive benefit of SRL on the development of transplant vasculopathy is a function of the degree of donor-recipient histoincompatibility and SRL doses.

Josep M. Campiaol

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Effect on Graft Vessel Disease Sirolimus Prevents Intimal Proliferation After Vascular Injury in Pigs

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Figure 2. Antiproliferative action of SRL on an experimental model of a mechanical lesion after vascular angioplasty. (Courtesy of Dr R. E. Morris, Stanford, Calif.)

SRL---A Nonnephrotoxic Drug The main side effect of calcineurin inhibitors is nephrotoxicity, directly related to their mechanism of action and recently suggested to be a risk factor for the development of CAN. SRL does not inhibit calcineurin; thus it is anticipated that SILL v'ill lack the acute nephrotoxicity profile of tile calcineurin inhibitors. There is much evidence in experimental models indicating that SRL does not have the nephrotoxicity associated with calcineurin inhibitors. In a rat model, it has been sho~al that SRL does not induce nephrotoxicity at doses 3 times higher than its effective immunosuppressive doses.38 In addition, infusion of a therapeutic dose of SRL had no deleterious effects on renal function in pigsP 9 In a more recent report, it was shown that only calcineurin inhibitors resulted in glomerular dysfunction in an acute experimental rat model of nephrotoxicity?° A pilot study in patients with psoriasis showed a strictly normal renal function with a progressive increase in the doses of SRL (Fig 3)N Phase I and II clinical trials have shown that SRL alone did not impair renal function or glomerular filtration rate. 4~14 The phase H European studies comparing SRL with cyclosporine showed a completely different safety profile between the 2 drugs, especially in terms of renal function.43,+~ Patients treated with SRL had significantly better renal function than patients treated with cyclosporine at 6, 12, and 24 months after renal transplantation and better control of blood pressure.

Also, other renal parameters such as uric acid, phosphorus, magnesium, and potassium were within norreal limits in patients treated with SRL. a3-~5 The lack of nephrotoxicity associated with SRL could be decisive for tile treatment and prevention of CAN. There are many clinical studies showing the relevance of serum creatinine at 3, 6, and 12 months after renal transplantation as a positive and strong predictor for the development of CAN.

SRL-Based Therapy Two European phase II studies with SRL used as the base therapy compared with cyclosporine showed similar results in terms of acute rejection and patient and graft survival. 43,'H Graft and patient survival were similar between the 2 groups at 12 and 24 months, without statistical differences. The incidence and severity of acute rejection also were similar between the 2 gToups, showing that SRL is equipotent to cyclosporine, ahhough tile safety profile was very different between the 2 immunosuppressive drugs. In terms of renal function, serum creatinine and calculated glomerular filtration rate were significantly better in patients treated with SRL compared with those treated with cyclosporine at 3, 6, 12, and 24 months after renal transplantation (Fig 4). Moreover, the control of blood pressure was also significantly better in patients treated with SRL in terms of blood pressure measurements and the number of antilwpertensive drugs. Although this experience

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groups, using calcineurin inhibitor-free immunosuppressive regimens with SRL, have shown a better renal profile than that observed in patients treated with cyclosporine or tacrolimus, with a very low incidence of acute graft rejection. 4Ga7

represents pilot and limited studies, as well as the first time that SRL was used as the base therapy, the results are very positive in terms of graft and patient survival, incidence of acute rejection, and renal function. 43~5 In fact, more recent studies from different

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The better renal function obtained in patients treated with SRL as base therapy may predict a lower incidence of CAN, with a significant improvement in graft survival. Moreover, the improvement in blood pressure also could be a positive factor for the prevention of cardiovascular mortality.

Cyclosporine Withdrawal Studies With SRL Two recent studies (phase II and Ill) showed that SRL allows an early and successful withdrawal of cyclosporine in renal transplantation, resulting in improved renal function and lower blood pressure. 48,49 Both studies had similar objectives with slightly different designs. In the phase ffl study, all patients started with a triple therapy of cyclosporine plus SRL plus steroids, and at 3 months after transplantation, patients were randomly assigned to cyclosporine withdrawal or to continuation of the same triple therapy. The withdrawal of cyclosporine was associated with a slight increase in the incidence of acute rejection, although most episodes were grade I (Banff classification) and successfully treated with methylprednisolone. Graft and patient survival were similar between the 2 therapeutic groups at 12 months, although at 24 months it appeared that graft survival was slightly better in the cyclosporine withdrawal group. Renal function was significantly

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Figure 5. Improvement of renal function (glomerular fih,ation rate, GFR) after cyclosporine (CsA) withdrawal with the usc of SRL at 24 months after renal transplantation. Aslerisk, P < .05. Note: At 24 months, morc paticnts in the SRL group expericnced an improvement in glomerular fihration rate after randomization (75% vs 30%,P < .001).

better after cyclosporine withdrawal, with a progressive improvement in the serum creatinine level in that group of patients (Fig 5). 4~ Two years after transplantation, serum creatinine was almost 50% lower and the glomerular filtration rate was 15-mL/ min higher in the SRL group. Also, blood pressure was significantly better after cyclosporine withdrawal, with lower systolic and diastolic blood pressures. Preliminary data oil renal biopsies performed at I year showed that the progression to chronic lesions (interstitial fibrosis, glomerulosclerosis, and transplant vasculopathy) was significantly reduced in the SRL group• Tiffs trial could be the first study to show that the elimination of cyclosporine with the use of SRL is associated with a lower incidence of CAN. Results at 3 and 5 years need to confirm this hypothesis. 4s

Conversion From Calcineurin Inhibitors to SRL Recently, a number of studies investigating the potential for conversion of long-term transplant patients fi'om a calcirleurin inhibitor-based regimen to SRL-based treatment have appeared in the literature, primarily involving patients with either CAN or chronic calcineurin-inhibitor nephrotoxicity (or both). 5°'53 These studies have shown that conversion

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can be accomplished successfully and long-term renal transplant patients can be maintained on an SRL, calcineurin inhibitor-free regimen with a favorable outcome, particularly with respect to kidney function, blood pressure, and glucose tolerance. A variety of protocols for converting patients from a cyclosporine-based to an SRL-based regimen appear to have been used with considerable success. Dominguez et al -~° from Halifax, Nova Scotia, Canada, showed the improvement in renal function after withdrawal of cyclosporine or tacrolimus in 20 transplant patients treated with calcineurin inhibitors. 5n In our unit, during the last 2 years we have converted more than 50 patients with either CAN or chronic nephrotoxicity, or both, from cyclosporine to SRL with considerable success. In terms of renal function, 75% of the converted patients experienced a marked improvement in serum creatinine level and creatinine clearance, with a better control of blood pressure. In some patients, mild proteinuria developed after withdrawal of cyclosporine, probably related to the glomerular hemodynamic changes induced by the cessation of cyclosporine. Other experiences from Diekmann et al -~1 and Sindhi et al r'~have shown similar results, with a significant improvement in renal function in most patients. "~'.'~2 The main risks of this conversion are overimmunosuppression and acute rcjection, although the incidence of acute rejection was negligible after SRL conversion in all these experiences. How quickly withdrawal of cyclosporine therapy is attempted may be dependent on the individual patient and the experience of the group. Because SRL has been shown to be equal to cyclosporine in terms of immunosuppressive efficacy, the concept of immediate cessation of cyclosporine and replacement with SRL is possible. For patients wbo are not in an immediate posttransplant phase, the risk of acute rejection does not appear to be particularly high fi'om experiences to date. Indeed, the possibility of overimmunosuppression, with its accompanying risks of infection or malignancy, should be considered when patients are to be converted from cyclosporine to SRL. There also may be patients in whom an immediate withdrawal of cyclosporine is required, such as those with symptoms of hemolytic-uremic s)qadrome or when other side effects require cessation of cyclosporine. However, when risk of acute rejection is a consideration, perhaps in the early posttransplant period, an approach in wbich a tapered reduction of cyclosporine is used may be considered.

17

The timing of conversion to SRL is important. In principle, patients may be converted from their existing cyclosporine-based immunosuppressive regimen to an SRL-based regimen at any time after transplantation. However, recent studies have provided clear evidence that, for patients showing signs of nephrotoxicity or CAN, conversion when patients are exhibiting early signs of renal dysfunction is considerably more likely to lead to a successful outcome compared with conversion ofpatients in whom nephrotoxicity has progressed to the chronic stage. Therefore conversion should be considered for renal transplant patients as soon as early signs of nephrotoxicity or CAN are observed (eg, creatinine levels above 150 /xmol/L with histologic confirmation). The ideal time to initiate conversion is before signs of frank renal damage are observed.

Summary CAN represents the primary cause of gTaft loss after the first year of renal transplantation. Calcineurininhibitor nephrotoxicity may be one of the most important pathogenetic factors in the development of this complex entity. SRL is a new immunosuppressive drug, with a mechanism of action that is different from that of caleineurin inhibitors and without their nephrotoxicity. SRL blocks the mTOR protein, which is a central point on the control cell cycle, through protein synthesis and transduction processes. Moreover, SRL has potent antiproliferative activity shown in different clinical and experimental models. Today, there is sufficient evidence to conclude that SRL could be a beneficial drug in the treatment and prevention of CAN. Renal function is significantly better in patients treated with ca[cineurin inhibitor-free immunosuppressive therapy, and the withdrawal of cyclosporine is associated with a better chronicity of renal lesions. Three-year renal allograft biopsies from patients in the cyclosporine withdrawal study need to confirm the hypothesis that the incidence of CAN is significantly lower with SRL.

References I. Tilney NL, Milford EL, AraujoJL, et al: 'Experiencewith cyclosporine and steroids in clinical renal transplantation. Ann Sm'g 1984,200:605 2. FergusonRM, SommerBG: Cyclosporinein renal transplantation: a single institt,tional experience.Am .J Kidney Dis 1985,5:296

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Josep M. Campistol

3. Roth D, ColonaJ, Burke GW, el al: Primary imrnunosuppression with tacrolimus and mycophenolate mofetil for renal ,'dlograft recipients. Transplantation 1998, 65:248 4. Miller J, Mendez R, Pirsch JD, et al: Safely and efficacy of tacrollmus in combination ~4ih mycophenolate mnt~iil (M~.IF) in cadaveric renal transplant recipients. FKS06A,IMI: Dose-Ranging Kidney Transplant Stud)' Gronp. Transplantation 2000, 69:875 5. Hariharan S, Johnson CP, Bresnahan BA, el al: hnproved graft snrvival after renal transplantation in the United States, 1988 Io 1996. N EnglJ Med 2000, 3'12:605 6. CeckaJM: The UNOS Scientific Renal Transplant Regist~'2000. Clin Transpl 2000, 18:1 7. Gjertson DW, CeckaJM: Mortality's role in kidney transplant failures. Transplant Proc 2001, 33:1212 8. Wallet" JR, Nicholson ML: Molecttlar mechanisms of renal allograft fibrosis. Br.J Surg 2001, 88:1-1-29 9. Womer KL, VellaJP, Sayegh MI-[: Chronic allograft d)~thnclion: mechanisms and new approaclws Io therapy. Semin Nephrol 2000. 20:126 10. Vazquez MA: Chronic rejcciion of renal transplants: new clinical insights. Am.J Mcd Sci 2000, 320:43 I I. Pascual M, Swinlbrd RD, IngelfingerJR, ct al: Chronic rejection and chronic Q'closl~x'in toxicity in remd allografls. Immunol Tt×lay 1998, 19:514 12. Campistol JM, Grinyo JM: Exploring treatment options in renal transplantation: The prohlems of chronic al[ograft dysfunction and drug-related nephrotoxicity. Transplantation 2001, 71:SS42 (supp[) 13. Hariharan S: Long-term kidney transplant survival. Am J Kidney Dis 2001, 38:$44 (Snppl) 14. Racusen LC, Solez K, CoMn R: Fihrosis and atrnphy in the renal allografi: interim reporl and new direclions. Am J Transplant 2002, 2:203 15. Pascual M, Williams WW, Cosimi AB, et al: Chronic renal allografi d)~function: a role Ibr myeophenolatc mofi:til? Transplantation 2000, 69:1749 16. Kasiskc BL: Epidemiology of cardiovascuhn" disease alter renal transplantation. Transplantation 2001, 72:$5 (Suppl) 17. Massy 73\: Cardiovascular risk factors in kklney Iransplantation. Curr Opin Urol 2001, 11:139 18. Pascual M, Tberuvath T, Kawai T, el al: Strategies to improve long-term outcomes after renal transplantation. N EnglJ Mcd 2002, 346:580 19. Baker H, Sidorowicz A, Sehgal SN, et al: Rapamycin (AY22,989), a new antifungal antihiotic. HI. In vitro and in vivo evaluation.J Antibiot (Tokyo) 1978, 31:539 20. Sehgal SN: Rapamune (Sirolimus, rapamycin): An overx4ew and mechanism of action. Ther Drug Monit 1995, 17:660 21. Dumont FJ, Staruch MJ, Koprak SL, et al: The immunosuppressive and toxic effects of FK-506 are mechanistically related: pharmacology of a novel antagonist of FK-506 and rapamycin. J Exp Med 1992, 176:751 22. Venkataraman L, Burakoff SJ, Sen R: FK506 inhihits antigen receptor-mediated induction of c-rel in B and T lymphoid cells.J Exp Med 1995, 181:1091 23. Kuo CJ, ChungJ, Fiorentino DF, et al: Rapamycin selectively inhibits interleukin-2 activation of p70 $6 kinase. Nature 1992, 358:70 24. Fruman DA, Wood MA, Gjertson CK, et al: FK506 hinding protein 12 mediates sensitivity to both FK506 and rapamycin in routine mast cells. EurJ Immunol 1995, 25:563

25. Chen H, Luo I 1, Daloze P, el al: Long-term in vivo e0izcts of rapamycin on htmloral and cellular immune reslxmses in tile rat. Immtnmbiology 1993, 188:303 26. Gallo R, Padurean A,Jayaraman T, el al: Inhibition of intimal thickening after balloon angioplasly in porcine coronary arteries by targeting regadalors of the cell cycle. Circulation 1999, 99:2164 27. ]aster R, Bittorf T, Klinken SP, et al: Inhil~ition of prolifi'ralion hut UOI er)'th,'oid differentiation of J2E cells by rapai1Wcin. Biochem Pharmaco[ 1996, 51:[ 181 28. Manx SO, Jayaralnan T, Go 1,O, el al: Rapamycin-l:KBP inhibits cell cycle regulators of" proliferation in vascular smooth muscle cells. Circ Res [{)95, 76:412 29. Dumont 1':], Kastner C.b\: Translbrming gl'owd) lactor beta I inhihits inteHeukin-l-induced but enhances ionomycin-indueed intcrl{won-ganlma pr~lttclion in a T cell lynll)honla: Comparisnn with tile ellbcts ofrapamyein.J Cell Physiol 19{)-t, 160:141 30. Tcrada N, Patel HR, Takase K, el al: Rapanlychl selectively inhibits translation o[" m|~z\As cntx.~iing clongalion 1actors and ribosonlal ln'oichls. Proc iNall Acad ,Sci U S A 1994, 91:11477 ] l . |~iv,'anlala S, Sakaida H, Hori T, eta[: The ttprcgulalion nf p27Kipl hy rapanlycin rcsuhs in O l arrcsi in exponentially gl"o~4llg T-cell lines. Bloc×l 19{18,91:561 32. Cooper MH, Gregory SH, StaFzl TE, el al: Rapamycin but not FK506 inhibits the proliferation of mononuclear pimgo~Ttes induced by colony-stinmhlting filctors. Transphmiatkal 199-I, 57:433 33. Grego,'y CR, Huie P, Billinglmm ME, ct al: Rapamycin inhihits arterial intimal thickening caused by both alloimmunc and mechanical injury. Its cf['cct oll cellular, growth 1actor, and q,lokinc response in ittjurcd vessels. Transplantation 1{193, 55:1409 34. Sousa .]E, Costa MA, Abizaid A, el al: lmck of neointinud proliferatkm alier implantation of sirolimus-cnatcd stcnts in human coronary arteries: A qnantitativc coronary angiography and three-xlinlensional intravascttlar uhrasotmd study. Circulation 2001, 103:192 35. Morice MC, Serruys PW, SousaJl'], el al, flit"the RAVEL Study Groulx Randomized study with the sirolimus-coated Bx vclocit), balloon-expandable stent in the treatment ofpaticnls with de novo native corollln'y arter), lesions. A randonlized conll)al'ison of a sirolimus-clnting stem with a standard stcnt lbr coronary revascularizatkm. N EnglJ Mcd 2002, 346:1773 36. Gcerling Ib\, de Bruin RW, Scheringa hi, et al: Suppression of acute rejection prevents graft arteriosclerosis after allogcneic aorta transplantation in the rat. Transplantation 199-k 58: 1258

37. Meiser BM, Billingham ME, Morris RE: EIl~cts ofcyclosporin, FK506, and rapam)vin on graft-vessel disease. Imncel 1991, 338:1297 38. DiJosephJF, Sharnla RN, ChangJY: The effect of rapamycin on kidney Ihnction in the Sprag'ue-Dawley rat. Transplantation 1992, 53:507 39. Andoh TF, Burdmann EA, Fransechini N, et al: Comparison of acute r,'ipamycin nephrotoxicity with c3,closlxwinc and FK506. Kidney hit 1996, 50:1110 40. Yoshimnra R, Yoshimura N, Ohyama A, et al: The effcct of irnmunosuppressive agents (FK-506, rapanwcin ) on rcnal P450 s)~tcms in rat models.J Pharm Pharmacol 1999, 51:941 41. Reitamo S, Spuls P, Sassolas B, et al, for the Sirolimus F,uro-

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pean Psoriasis Study Group: Efficacy ofsirolimus (rapamycin) administered concomitantly with a subtherapeutic dose of cTclosporin in the treatment of severe psoriasis: A randomized controlled trial. Br.] Dermatol 2001, 145:438 42. Meier-l~'iesehe HU, l~q)lan B: Toxicity and efficacy of sirolimus: Relationship to whole-bltxxl concentrations. Clin Ther 2000,22:B93 (Suppl) ,I-3. Krcis H, CisterneJM, l.amd W, ct al: Sirolimus in association with mycophenolate mofetil inductitm for the prevention of acute graft rejection in n'enal allograft recipients. Transplantation 2000, 69:1252 44. Groth CG, Backman L, MoralesJM, et al: Sirolimus (rapamycin)-based therapy in hunaan renal transplantation: similar effica~' and different toxicity compared with c3,closl~rine. Sirolimus European Renal Transplant Study Group. Transplantation 1999, 67:1036 45. Morales JM, Wramner L, Kreis H, et al, and the Sirolimus European Renal Transplant Study Group: Sirolimus does not exhibit nephrotoxicity compared to cyclosl~rine in renal transplant recipients. AmJ Transplant 2002, 2:436 46. Sirolimus and nLveophenolatc rnnfetil for calcineurin-free innmuriosuppression in renal transplant recipients. AmJ Kidney Dis 2001, 38:SI6 (Suppl) 47. HongJC, Kahan BD: A calcineurin antagonist-free induction

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strategy lbr immunosuppression in cadaveric kidney transplant recipients at risk for delayed graft function. Transplantation 2001, 71:1320 48. Johnson RW, Kreis H, Oberbauer R, et at: Sirolimus allows eaHy cyclosporine withdrawal in renal transplantation resulting in improved renal function and lower blood pressure. Transplantation 2001, 72:777 49. Gonwa T, Hricik DE, Brinker K, et al, for the Sirolimus Renal Function Study Group: Improved renal thnction in Sirolomustreated renal transplant patients following early cyelosporine elimination. Transplantation (in press) 50. Dominguez J, Mahalati K, Kiberd B, et ah Conversion to rapamycin immunosuppression in renal transplant recipients: report of an initial experience. Transplantation 2000, 70:1244 51. Diekmann F, Waisex'J, Fritsche L, et ah Conversion to rapanwcin in renal allograft recipients with biopsy-proven calcineurin inhibitor-induced nephrotoxicity. Transplant Proc 2001, 33:3234 52. Sindhi R, Webber S, Venkataranmnan R, et ah Sirolimus for rescue ancl primary immunosuppression in transplanted children receiving tacrolimus. Transplantation 2001, 72:851 53. Nishkla S, Pinna A, Verzaro R, et al: Sirolimus (rapamyein)based rescue treatment following chronic rejection after liver transplantation. Transphmt Proc 2001, 33:1495