Sirolimus therapy without calcineurin inhibitors: Necker Hospital 8-year experience

Sirolimus Therapy Without Calcineurin Inhibitors: Necker Hospital 8-Year Experience E. Morelon and H. Kreis

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INCE THE FIRST reports of the preclinical results obtained with sirolimus showing that it had potent immunosuppressive activity, we have helped to develop it as a competitor of calcineurin inhibitors (CNI) and not as an add-on agent. In animals, sirolimus has exhibited potent antirejection activities as well as the ability to prolong graft survival.1 Its mechanism of action was shown to be distinct from that of cyclosporine (CsA) and tacrolimus. Because the significant nephrotoxicity, neurotoxicity, and hypertension associated with CsA and tacrolimus are partly due to calcineurin blockade,2– 4 sirolimus could be expected to have a different toxicity profile, and thus be the ideal alternative to CNI. We therefore participated in many trials aimed at either replacing or completely avoiding CsA. The formers were either phase III or compassionate trials, whereas the latter were trials of sirolimus-based phase II protocols. Phase III and II multicenter protocols have already been published5– 8 and will not be reported here. We will only deal with our personal experience on the use of sirolimus. This includes both the switch from CNI to sirolimus and the use of sirolimus as base therapy. However, none of our data are derived from randomized studies. They only concern the results obtained when the data acquired for various protocols were applied to everyday practice. SWITCH FROM CALCINEURIN INHIBITORS TO SIROLIMUS Different Protocols for Switching

The absence of nephrotoxicity of sirolimus, coupled with its immunosuppressive power, make it a strong candidate for eventually replacing CNI. Sirolimus can be introduced, either right from the start of transplantation as base therapy, or during the posttransplantation follow-up. Various protocols for switching from CNI to sirolimus have already been tested over the past few years. In Study 310, patients were treated for 3 months with steroids, sirolimus, and CsA. Thereafter, they were randomly allocated either to continue with the same tritherapy (arm A) or to change to a bitherapy regimen consisting of sirolimus and steroids (arm B). In the latter case, sirolimus doses were raised to reach a trough level of 20 –30 ng/mL measured by high performance liquid chromatography (HPLC) while CsA was tapered to 0 over a 1-month period. The results of this study 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(03)00244-6 52S

are reported elsewhere in this issue and have already been published.8 Study 316 is an ongoing study on switching from CNI to sirolimus in patients with biopsy-proven chronic allograft nephropathy who were transplanted from 6 months to 5 years prior to randomization. These patients have been randomly allocated to stay on CNI or to receive sirolimus. The main end-point is renal function 1 and 3 years after randomization and the course of chronic allograft nephropathy is being evaluated on protocol biopsies. This study is still under way and the results are not yet available. Protocol 501 is a compassionate protocol facilitating the procurement of sirolimus, which had not yet been licensed at the time of the study. When enrolled in the protocol most patients had chronic transplant nephropathy with evidence of CNI nephrotoxicity on renal biopsy. Therefore, in these cases sirolimus was introduced and CNI withdrawn, without transplant loss. In other cases, sirolimus was introduced early to avoid CNI nephrotoxicity in patients with a marginal kidney or following severe CNI side effects such as thrombotic microangiopathy. The results obtained by our group with this protocol are reported below. Results for Protocol 501

Protocol 501 is an open, nonrandomized compassionate protocol allowing a switch from CNI to sirolimus. Unfortunately, the absence of a control group made it impossible to obtain reliable results for the efficacy of the switch. However, this protocol did highlight the safety of sirolimus. Some of the resulting side effects had already been reported in previously published multicenter studies, but others, which had not yet been reported, were shown by analysis of the data obtained with this protocol. Various Ways of Switching in Protocol 501. Switching from CNI to sirolimus can be done either in a stepwise process or instantaneously. When CsA and sirolimus are given simultaneously the area under the curve (AUC) of sirolimus is increased.9 It is therefore important to increase From the Service de Transplantation et de Re´animation, Hoˆpital Necker, Paris, France Address reprint requests to Dr H. Kreis, Service de Transplantation et de Re´animation, Hoˆpital Necker, 149 rue de Se`vres, 75015 Paris, France. E-mail: [email protected] © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35 (Suppl 3A), 52S–57S (2003)

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the sirolimus doses when eliminating CsA to avoid low sirolimus blood trough levels. Switching from CsA to sirolimus can be done over a 4-week period, as in protocol 310. In that case, 2 mg/d sirolimus is the starting dose, and CsA is tapered down by 25% every week. Blood sirolimus levels are monitored every 5 days and its daily dose is adjusted every week until satisfactory trough levels are obtained. The positive aspect of this stepwise switching is that sirolimus blood trough levels can be monitored to avoid harmful underimmunosuppression in the initial posttransplant period. Furthermore, gradually raising sirolimus doses makes it easier to prevent dose-related side effects. However, this stepwise type of switch increases the risk of a short period of overimmunosuppression. In most cases, this should not be harmful. However, we know that sirolimus increases CsA nephrotoxicity in a dose-dependent manner, and a transient increase in serum creatinine may occur during the overlapping period. In practice, a sudden switch from CNI to sirolimus without a transition period does not appear to cause any particular problem, except for cutaneous side effects, which we will report in greater detail below. Effectiveness of the Switch From CNI to Sirolimus in Protocol 501. Fifty kidney transplant patients were included in protocol 501 at the Necker Hospital in Paris (France) between 1997 and 2001. The indications for switching from CNI to sirolimus were chronic allograft nephropathy with probable CNI nephrotoxicity in 48 of these 50 patients, and thrombotic microangiopathy in the remaining 2. Thirty-two of the 50 patients experienced an initial improvement in renal function, as shown by a mean decrease in serum creatinine from 219 to 118 ␮mol/L. However, in 20 of these 32 cases, renal function again subsequently deteriorated. At the end of follow-up, it was defined by a mean serum creatinine of 160 ␮mol/L. The initial improvement might have resulted from the increase in renal perfusion that usually follows CNI withdrawal, and the subsequent deterioration in renal function probably reflects the progression of chronic alloantigen-dependent rejection that was not controlled by either CNI or sirolimus. The absence of a control arm in this compassionate protocol did not allow us to draw any conclusions about the long-term effectiveness of the CNI-sirolimus switch in kidney transplant recipients. Nevertheless, the results of controlled phase II trials, in which sirolimus was compared to CsA as base therapy, showed that at both 1 and 2 years, renal function was better in the sirolimus arm. It suggests that in the long run, switching from CNI to sirolimus should exert a beneficial effect by eliminating the nephrotoxic component of chronic allograft nephropathy. Safety. Lipid Disorders. Sirolimus-induced hyperlipidemia was found in 41 of the 50 patients. Hypercholesterolemia was present in 3 patients, hypertriglyceridemia in 10 others, and both were increased in the remaining 28. Hypercholesterolemia and hypertriglyceridemia were controlled by either statins or fibrates in 40 patients. Sirolimus had to be discontinued in only one patient due to ineffective antilipid treatment

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Hematologic Disorders. Anemia is probably the most severe sirolimus-induced hematologic side effect. It was observed in 36 of the 50 patients who switched to sirolimus. This anemia is aregenerative, with high ferritinemia and low serum iron. Its causes are probably multifactorial. In addition to sirolimus-induced toxicity, some degree of renal dysfunction and azathioprine or mycophenolate mofetil (MMF) medullar toxicity were frequently present, especially in patients who had been on azathioprine or MMF for many years. Sirolimus had to be discontinued in one patient because of persistent anemia. After sirolimus elimination, the hemoglobin level rose rapidly. Sirolimus can also induce thombocytopenia or leukopoenia, which are aggravated by the cumulative toxicity of DNA synthesis inhibitors. However, they were dose related, never severe, and sirolimus had to be stopped for only 2 out of 50 patients. Hepatotoxicity Increased liver enzyme levels can be observed in patients on sirolimus.8 This was the case in 2 of the 50 patients reported here, and was responsible for sirolimus cessation in one. Cholestatic hepatitis, which occurred after the introduction of sirolimus, was also observed in three patients. Liver biopsy, performed in one case, showed lesions suggestive of drug-related toxicity. After sirolimus cessation, the course of cholestatic hepatitis was reversed within 6 months. These data suggest that sirolimus may induce cholestatic hepatitis. Lung toxicity. In a randomized trial of the comparative effects of sirolimus and CsA, a higher incidence of pneumopathy was reported in patients given sirolimus.5 This pneumopathy was usually considered to be of infectious origin. Nineteen patients in the Protocol 501 series developed 25 pneumopathies; but an infectious origin could only be established for 13. The germs responsible for these pneumopathies were pneumococcus in two cases, hemophilus influenzae in one, legionella pneumophilla in two, mycobacteria xenopia in two, Pneumocystosis carinii in one, Staphylococcus aureus in one, and Staphylococcus epidermidis in one; three cases were of unknown origin. The remaining 12 patients exhibited bilateral interstitial pneumopathy with no identifiable infectious cause, despite an extensive search. For this reason, and because of their fast improvement after sirolimus withdrawal, these patients’ pneumopathies were considered to be sirolimus related. A clinical description of the first eight sirolimus-linked cases of pneumopathy has already been reported.10 Most are described as bilateral interstitial pneumopathy predominantly targeting both lung bases and extending to the periphery. Clinical signs are nonspecific and include dry cough, dyspnea, fever, and fatigue. Interestingly, one patient developed bilateral interstitial pneumopathy, evidenced on protocol chest x-rays, but remained asymptomatic throughout the course of the disease. Usually, bronchoalveolar lavage (BAL) revealed lymphocytic pneumopathy, with the predominance of activated CD4⫹ lymphocytes. In two cases, BAL showed intra-alveolar hemorrhage. Transbronchial pulmonary biopsies, performed in

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two patients, showed features typical of bronchiolitis obliterans with organizing pneumopathy. Thus, the occurrence of an alveolar-interstitial pneumopathy in a transplanted patient on sirolimus requires a BAL. The absence of infectious agents and the presence of a large number of lymphocytes in the BAL fluid indicate a strong probability of pneumopathy due to sirolimus toxicity. After sirolimus withdrawal, clinical symptoms improved within 2 weeks, and chest x-ray showed a return to normal. Pulmonary function recovered completely within 3 months of sirolimus withdrawal. Similar recovery was obtained in two patients in our series just by decreasing sirolimus doses, and also in one patient reported by Mahalati et al.11 In a particularly severe case, 2 weeks of treatment with 1 mg/kg corticosteroids daily improved pulmonary function rapidly. The physiopathology of these pneumopathies remains unknown. However, they were mainly observed in patients treated with sirolimus as base therapy, with blood trough levels usually higher than 20 ng/mL. This suggests that sirolimus’ lung toxicity is dose related. Furthermore, the presence of activated CD4 lymphocytes in patients’ BAL suggests an immunologic mechanism. The presence of CD4⫹ lymphocytes producing IL-4 in patients’ blood might constitute a peripheral marker of the disease.12 Proteinuria. Switching from CNI to sirolimus was complicated by the development of proteinuria in 32 cases and a nephrotic syndrome in 18 cases. This proteinuria developed within the first weeks after the switch. Renal biopsies performed in 15 patients showed the presence of focal segmental glomerular sclerosis (FSGS) lesions in 5 cases, but revealed no informative changes in 7. FSGS lesions were not present in the biopsies taken before the switch. Furthermore, none of the 5 patients had FSGS as primitive nephropathy, which excludes the possibility that proteinuria recurred in the transplanted kidney. The role of sirolimus in the development of such proteinuria is not known. On the one hand, proteinuria was not reported, either in the randomized phase III trials in which sirolimus was compared to azathioprine13 or to a placebo,14 or in the phase II studies comparing the effects of sirolimus and CsA5,6; neither was it reported after an early switch from cyclosporine to sirolimus (Study 310).8 On the other hand, we know that 10% of patients on CNI have FSGS lesions on protocol biopsies.15,16 One explanation might be that CNI withdrawal leads to an increase in the renal blood flow responsible for the development of previously masked proteinuria in patients with preexisting CNI-related FSGS lesions. The description of the development of proteinuria in heart transplant recipients after CsA elimination because of its nephrotoxicity, without its replacement by sirolimus therapy argues in favor of this possibility.16 A significant decrease in proteinuria after the introduction of angiotensin-converting enzyme inhibitors in six patients also supports this hypothesis. However, it is also possible that sirolimus might have a direct toxic effect on the glomeruli previously injured by CNI treatment, as reported in experimental models.17 An ongoing randomized trial of the switch from CNI to siroli-

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mus that includes pre- and postswitch biopsies should allow better evaluation of the actual incidence of this proteinuria. Miscellaneous. Dermatologic lesions: Sirolimus therapy is often complicated by the early development of mouth ulcers, which are often numerous, large, and persist for a few weeks. These ulcers developed in 13 of our 50 patients and lasted several weeks. Usually, they do not recur and can be treated symptomatically using sucralfate-based mouthwash. One patient developed large widespread mouth ulcers, which led to sirolimus withdrawal. Sirolimus treatment can also be responsible for cutaneous lesions, which are often referred to in the literature as acnea lesions. However, unlike acnea lesions, these skin lesions are usually pustulous, monomorphic, and florid, and may appear on the scalp, face, and trunk. They were only observed in 10 male patients, and not in female patients. No germs could be found on the cutaneous biopsies performed in five patients. These skin lesions disappeared spontaneously within 4 – 6 weeks and did not usually recur. Their pathophysiology is still unknown. Edema and Algodystrophy: Another side effect of sirolimus is the development of lower-limb edemas, observed in 13 patients. They were often painful and sometimes inflammatory. They do not appear to be dose related and often persist despite diuretic treatment that bring on an only partial improvement. Lastly, bone pain suggestive of algodystrophy were also observed in three sirolimus-treated patients, predominantly in the ankles, shins, and knees. Their intensity was increased by activity. Bone scintigraphy showed areas suggestive of hyperfixation at the sites of pain. Conclusions about switching. Our clinical experience shows that the side effects of sirolimus are more critical, both in number and severity, when sirolimus replaces CNI in previously immunosuppressed patients than when it is given as base therapy from the start of grafting. The increased incidence of side effects mainly concerns mouth ulcers, skin lesions, pneumopathies, and anemia. The reasons for this increase are not known. The anti-inflammatory effect of high-dose steroids, as prescribed at the start of the transplant procedure, might be one explanation. It seems that the later sirolimus is used, the higher the incidence of its side effects. This suggests that sirolimus toxicity is higher when patients are already weakened by prolonged immunosuppressive treatment. The advantage of switching from CNI to sirolimus and the best period for it remain to be demonstrated by the randomized trial 316 currently in progress. However, to avoid CNI nephrotoxicity, it is preferable to implement the switch as early as possible. There is indeed little to expect from such replacement when the transplant already exhibits irreversible lesions of chronic allograft nephropathy. Furthermore, our experience shows that sirolimus is better tolerated when it is prescribed in the immediate posttransplant period than when it is introduced later.

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SIROLIMUS-BASED PROTOCOLS

Preclinical data with sirolimus, as well as the initial phase II and III multicenter studies, demonstrated that sirolimus was in fact a potent immunosuppressive agent. The European approach was then to challenge sirolimus against CsA to demonstrate that it might be possible to avoid CNI. This possibility was appraised in two phase II trials in which sirolimus was compared directly with CsA for the prevention of acute rejection in renal allograft recipients. Patients were treated with either sirolimus or CsA in combination, in both cases, with steroids and either azathioprine or MMF.5,6 Sirolimus was shown to be as effective as CsA in preventing acute graft rejection, with a rejection rate of 35% versus 29% for CsA. This study also demonstrated that sirolimus had a safety profile without nephrotoxicity, different from that of CsA. Renal function returned to normal in the patients given sirolimus as base therapy, whereas it gradually deteriorated in those on CsAs-based therapy. These results were still evident after a 5-year follow-up. There were other favorable findings for sirolimus with regard to hypertension and tremor. It was, however, associated with hyperlipidemia and thromboleukopenia. Remarkably, although the number of patients was not very large, no cancers were observed in the patients given sirolimus, whereas they occurred in 5% of those given CsA. Sirolimus-Based Protocols Plus Biological Induction

Rationale. Because the overall results reported for sirolimus demonstrated its efficacy and freedom from nephrotoxicity, we decided to design a sirolimus-based regimen as regular therapy for our renal transplant recipients. However, because the incidence of acute rejection observed during the phase II studies was considered a little too high, we added an induction treatment with basiliximab to the previous sirolimus, MMF, and prednisolone protocol. The confirmation that an induction period could effectively reduced the rejection rate had already been reported by Flechner et al.,7 who were thus able to reduce the acute rejection incidence to less than 10%. However, their sirolimus trough levels were probably a little too low. Protocol. Since March 1, 2002, all recipients of a cadaver kidney not enrolled in a research protocol recipients in our department were given a therapeutic regimen combining sirolimus, starting with a loading dose of 20 mg, followed by a daily morning dose of 8 mg, and then adjusted so as to maintain blood trough levels at 12–20 ng/mL (as measured by HPLC-UV); MMF, at a dose of 750 mg BID; and 3 prednisolone, at a dose of 1 mg/kg for 10 days rapidly tapered to 0.25 mg/kg at day 30 and 0.15 mg/kg at 6 months, and maintained thereafter. An induction treatment consisting of the infusion of 20 mg basiliximab on days 0 and 4 was always added to this regimen. Protocol biopsies were scheduled for all patients on the day of transplantation, and 3 months, 2 years, and 10 years thereafter.

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Demography. Up to now, 27 patients have had the above treatment. We report here the first results obtained from 17 patients after 1–9 months of follow-up. They comprised nine men (53%) and eight women (47%). Mean recipient age was 45 ⫾ 11 years, and mean donor age, 40 ⫾ 11 years. Three patients received a kidney from a living-related donor, 13 received a first cadaver kidney, and one received a second cadaver kidney. The mean ischemia time was 18 ⫾ 8 hours. Eleven patients (64%) received a kidney from a cadaver donor whose death was due to a cerebrovascular accident. Anti-HLA antibodies were present in three patients, including one with antibodies directed at 96% of the panel. In six patients (35%), the original disease was chronic glomerulonephritis, including one patient with focal glomerulosclerosis. Results. The incidence of delayed graft function (DGF) was relatively high (11/17 patients, 65%). This may have been due to the large proportion of patients (11/17) who received a kidney from a marginal donor. Despite this, the incidence of acute rejection was remarkably low; only one patient (5.8%) had biopsy-proven acute rejection. This patient had received a kidney from his haplo-identical brother. In addition to this low incidence of acute rejection, renal function gradually returned to normal, and at 2 months, mean serum creatinine was 123 ⫾ 19 and remained at the same level for another 4 months. For as yet unexplained reasons, significant proteinuria (mean 2.73 g on day 1, and 2.53 g on day 7) was observed in 14 of the 17 patients. However, this proteinuria progressively decreased to a mean value of 0.33 g/d at 4 months. With the regimen initially designed it was difficult to obtain satisfactory sirolimus trough levels rapidly. Therefore, most patients were underimmunosuppressed during the first 2 weeks, as shown in Fig 1. To improve this, we recently altered the initial sirolimus dose schedule by giving a daily dose of 12 mg instead of the original 8, after the loading dose of 20 mg. Safety. It is still too early to determine the incidence of the side effects due to this treatment regimen. However, none of the 17 patients had a serum cholesterol level above 6 ␮mol/L (Fig 2A), and only 5 (29.4%) were given antilipid agents. Triglyceride levels were only slightly above normal (Fig 2B). Anemia, thrombopenia, and leukopenia were observed in 11, 9, and 5 patients, respectively. Biological disorders were mostly hyperglycemia, observed in three patients (18%), increased transaminases in two (12%), hypocalcemia in one (6%), and hypophosphoremia in one (6%). In 10 patients (58%), no biological disorder was observed. Surgical complications were delayed wound healing, urinary leak, lymphocele, and wound hematoma in one patient each. The remaining 13 patients (76%) had no surgical complications. Infections were observed in 11 of the 17 patients (66%). Mouth ulcers were among the most frequent (3 patients, 18%); a wound abcess occurred in 2 (12%), urinary tract infection, in one (6%) and Epstein-Barr virus replication in

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Fig 1.

Blood rapamycin trough levels during the first 6 months. Shaded area represent expected trough levels.

one (6%). Other types of infection accounted for the remaining 4 (24%). Other treatment-emergent complications are listed in Fig 3. They were mostly hypertension and diarrhea. Hypertension was observed in 13 of the 17 patients (76%), but all these patients were hypertensive at the time of transplantation. Diarrhea occurred in nine patients, but they were also on MMF. Although it is impossible to draw any conclusions from this study, the first results are as expected. They confirm that sirolimus is effective in preventing acute renal graft rejection, as reported by Flechner et al.,7 and also show that it is not toxic to renal function. It is even remarkable that in this small series of patients, who mostly received kidneys from marginal donors, renal function returned to normal between months 2 and 6. The fact that, despite these drawbacks, sirolimus was effective in preventing acute rejection suggests that it could be successfully used with marginal kidneys.

Fig 2.

The reason for the proteinuria observed is not yet understood. It is probably not sirolimus related because it decreased with time, whereas the reverse would have been the case if it had been caused by sirolimus. It might be related to the poor quality of the transplanted kidneys and the high incidence of DGF. Our data also show that therapeutic drug monitoring (TDM) is mandatory with sirolimus because of the great interindividual variability of the dose/trough level ratio. Initially, low blood sirolimus levels may be responsible for acute rejection, but no complications have been attributed to a short period of excessively high levels. As the first levels cannot usually be obtained until 7– 8 days after transplantation, it seems appropriate to start the treatment with a relatively high dose of sirolimus to avoid initial underimmunosuppression. Our present schedule, which is a 20-mg loading dose followed by a 12-mg daily dose until the first trough level measurement can be obtained, seems satisfactory.

Lipid changes. (A) Cholesterol. (B) Triglycerides.

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Fig 3.

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Other treatment emergent sides effects.

CONCLUSION

Our experience with sirolimus since 1995 has now convinced us that this agent can replace CNI, either in the course of follow-up or better, as base therapy, starting just after the operative procedure. All sirolimus side effects are not yet known, but they seem to be different from those observed with other immunosuppressive agents. They are dose related and their incidence seems to have decreased since the initial trials, in which much higher trough levels were used. It is still necessary to wait a few more years to establish the precise impact of sirolimus on long-term graft function, chronic allograft vasculopathy, and cancers. REFERENCES 1. Sehgal S: Ther Drug Monit 17:660, 1995 2. Bennett WM, De Mattos A, Meyer MM, et al: Kidney Int 50:1089, 1996 3. Andoh TF, Burdmann EA, Bennett WM: Semin Nephrol 17:34, 1997

4. Mayer AD, Dmitrewski J, Squifflet JP, et al: Transplantation 64:436, 1997 5. Groth CG, Ba¨ckman L, Morales J-M, et al: Transplantation 67:1036, 1999 6. Kreis H, Cisterne JM, Land W, et al: Transplantation 69:1252, 2000 7. Flechner SM, Goldfarb D, Modlin C, et al: Transplantation 74:1070, 2002 8. Johnson RWG, Kreis H, Oberbauer R, et al: Transplantation 72:777, 2001 9. MacDonald A, Scarola J, Burke JT, et al: Clin Ther 22(suppl B):B101, 2000 10. Morelon E, Stern M, Israe¨l-Biet D, et al: Transplantation 72:787, 2001 11. Mahalati K, Murphy DM, West ML: Transplantation 69: 1531, 2000 12. Morelon E, Israe¨l-Biet D, Stern M, et al: Am J Transplant 2(suppl 3):204, 2002 13. Kahan BD: Lancet 356:194, 2000 14. MacDonald A: Transplantation 71:271, 2001 15. Takeda A, Uchida K, Haba T, et al: Clin Transplantation 15(suppl 5):22, 2001 16. Myers B, Sibley RK, Newton L, et al: Kidney Int 33:590, 1988 17. Daniel C, Ziswiler R, Frey B, et al: Exp Nephrol 8:52, 2000