Mini Review Sirolimus for GVHD Prophylaxis in Allogeneic Stem Cell Transplantation

Mini review Sirolimus for GVHD prophylaxis in allogeneic stem cell transplantation

C Cutler1,2 and JH Antin1,2

1Dana-Farber Cancer Institute, Boston MA, USA; and 2Brigham and Women’s Hospital, Boston MA, USA

Summary: Sirolimus: background and mechanism of action

Sirolimus is a novel macrolide immunosuppressant widely Sirolimus (Rapamunes, Wyeth) is a naturally occurring used in solid organ transplantation. We have conducted compound originally isolated from a soil saprophyte three clinical trials using this compound as prophylaxis (Streptomyces hygroscopicus) found uniquely on Easter against GVHD after allogeneic stem cell transplantation. Island (Rapa Nui). In addition to its immunosuppressive Our studies have demonstrated excellent GVHD control properties, sirolimus has antifungal, antiviral and anti- even when mismatched and unrelated donors were used. neoplastic properties. The morbidity and mortality associated with transplanta- Although structurally similar to other calcineurin in- tion were reduced due to the omission or reduction in hibitors (Figure 1), sirolimus binds uniquely to FK binding methotrexate dose. Furthermore, CMV reactivation and protein 12 (FKBP12) and then complexes with mammalian fungal infection rates were low. However, we have noted target of rapamycin (mTOR). Sirolimus does not interact that sirolimus may be associated with increased rates of with calcineurin or its downstream effectors. As shown in thrombotic microangiopathy after transplantation. Siroli- Figure 2, the sirolimus-FKBP12-mTOR complex inhibits mus has other uses, such as the treatment of established IL-2-mediated proliferation signaling via several distinct acute and chronic GVHD, and may be useful for biochemical pathways, with a reduction in DNA transcrip- treatment of post transplant lymphoproliferative disorder tion, DNA translation, protein synthesis and cell cycling, and perhaps as an antineoplastic agent against a wide ultimately leading to T-cell immunosuppression. Upstream variety of hematologic and solid neoplasms. pathways that interact with mTOR include the PTEN/PI3 Bone Marrow Transplantation (2004) 34, 471–476. kinase/Akt pathway and the Janus kinase pathway, which doi:10.1038/sj.bmt.1704604 is important in mediating IL-2-driven signaling from the Published online 26 July 2004 T-cell receptor.2 Although the mechanisms are less clear, Keywords: sirolimus; GVHD prophylaxis; allogeneic sirolimus appears to exert some of its immunosuppressive stem cell transplantation properties via inhibition of dendritic cell activity through a reduction in antigen uptake,3,4 cellular maturation,5 intracellular signaling6 and apoptosis induction in dendritic cells.7,8 Graft-vs-host disease (GVHD) remains the most signiﬁcant Sirolimus has been used widely in renal transplantation, cause of morbidity and mortality after allogeneic stem cell liver transplantation and in transplantation of other solid transplantation. Despite this, the combination of a organs. In renal transplantation, several randomized trials calcineurin inhibitor with methotrexate remains the stan- have demonstrated that the addition of sirolimus to an dard of care 15 years after Storb et al1 initially demon- established immunosuppressive regimen is associated with 9,10 strated its effectiveness. Numerous attempts to improve improved allograft survival and long-term renal func- 11 upon this regimen have been largely unsuccessful. In order tion after transplantation. Although sirolimus has not to address this problem, we began clinical trials with the been tested in a randomized fashion after liver transplanta- novel immunosuppressant, sirolimus, for GVHD prophy- tion, there is ample evidence to suggest that its introduction laxis after allogeneic stem cell transplantation in 2000. At to facilitate early calcineurin minimization has lead to a that time, the compound had already become a commonly reduction in adverse renal outcomes without increasing the 12–15 used agent in renal and hepatic transplantation. In this graft rejection rate after transplantation. review, we describe our experience with this compound and The use of combination immunosuppression with sir- discuss the potential future for the broader use of sirolimus olimus in stem cell transplantation is attractive, since the in stem cell transplantation. drug has nonoverlapping toxicities with the calcineurin agents and a different mechanism of action (Table 1). When used as part of combination immunosuppressive therapy, the combination of sirolimus with tacrolimus has been shown to be the most efﬁcacious. In vitro studies have Correspondence: Dr C Cutler, Dana-Farber Cancer Institute, 44 Binney St, D1B22 Boston, MA 02115, USA; suggested that this combination is more effective than E-mail: [email protected] sirolimus and cyclosporine in reduction of memory Published online 26 July 2004 T-cell production, apoptosis induction and cytokine Sirolimus in allogeneic transplantation C Cutler and JH Antin 472 Table 2 Clinical advantages and disadvantages of sirolimus

Advantages Disadvantages

Synergy with calcineurin No intravenous formulation inhibitors commercially available Nonoverlapping toxicity with Monitoring by HPLC necessary calcineurin inhibitors Long half-life Signiﬁcant drug interactions, particularly with antifungal agents Wide therapeutic window Myelosuppressive May potentiate thrombotic Figure 1 Structural comparison – sirolimus and tacrolimus. The structu- microangioapthy rally similar portion of the molecule is highlighted.

used in combination in comparison with sirolimus and cyclosporine.17,18

Practical aspects of sirolimus use in allogeneic transplantation Sirolimus is a relatively simple compound to use. Although it exists only as an oral formulation (liquid and tablet), due to its extremely long half-life (60–72 h), missed doses due to transplant-related nausea and mucositis are rarely of important clinical consequence. Drug levels must be measured by HPLC, as ELISA-based assays generally will measure both sirolimus and its metabolites, which are variably generated and variably immunosuppressive. Again, due to the long half-life of sirolimus, serum levels can be measured throughout the day, but trough measure- ments are preferred. Sirolimus has several known drug interactions, which largely resemble those of tacrolimus; Figure 2 Mechanism of action of sirolimus. Multiple downstream targets however, the concomitant use of voriconazole with of the sirolimus–FKBP12–mTOR complex are demonstrated, including sirolimus is difﬁcult, as drug absorption (as low as 10% those postulated to be critical to interactions within dendritic cells. at baseline) can rise as much as 100-fold due to inhibition of sirolimus metabolism by the cytochrome P450 enzyme, Table 1 Comparative proﬁle: sirolimus and calcineurin inhibitors CYP 3A4, in the bowel wall (Tables 1 and 2). Common side effects of sirolimus include the develop- Sirolimus Calcineurin inhibitors ment of hyperlipidemia (both hypercholesterolemia and FKBP binding FKBP12 only Multiple FKBPs hypertriglyceridemia) with prolonged use, particularly Molecular target mTOR Calcineurin when combined with a calcineurin inhibitor (cyclosporine Effects on cytokine Prevents multiple Prevents IL-2- more than tacrolimus). In addition, mild reversible signaling cytokine-stimulated stimulated cytokine cell cycling, including production cytopenias are common with prolonged exposure. Other IL-2 well-described side effects of chronic sirolimus use in the

Cell cycle arrest Prevents G1-S Prevents G0-G1 solid-organ transplant setting include lower extremity Dendritic cell activity Postulated edema, arthralgias, oral aphthous ulcers and interstitial k Antigen uptake pneumonitis.19 kCellular maturity None kIntracellular signaling mApoptosis Treatment of established GVHD Clearance Hepatic Hepatic Half-life Long (60–72 h) Intermediate (8–32 h) Predominant Mild Nephrotoxic In the only published report on therapy of established toxicities myelosuppression steroid-refractory acute GVHD, Benito et al described their Hyperlipidemia Neurotoxic experience treating 21 patients with sirolimus. Most Thrombotic Potentiation of Strong association patients were treated with 4–5 mg/m2/day and some were microangiopathy calcineurin effects given an oral loading of 15 mg/m2. Although the drug was active in this patient population (overall response rate 57%), the drug proved to be too toxic at the doses used, as production.16 When tested in a comparative fashion ﬁve patients developed thrombotic microangiopathy and after renal transplantation, early results suggested fewer many others had reversible cytopenias.20 episodes of acute rejection and better preservation of The efﬁcacy of this agent as therapy for chronic GVHD renal function when sirolimus and tacrolimus were in combination with calcineurin inhibitors has been

Bone Marrow Transplantation Sirolimus in allogeneic transplantation C Cutler and JH Antin 473 published in abstract format only. Johnston et al21 safe with the occurrence of only eight cases of veno- described a 56% overall response rate when sirolimus was occlusive disease of the liver (12%) and five cases of given with either tacrolimus or cyclosporine in patients with interstitial pneumonitis/idiopathic pneumonia syndrome established chronic GVHD, while Couriel et al22 described (8%). For the entire 65 patient cohort, chronic GVHD a 68% response rate to the combination of sirolimus and has been noted in 48% of patients surviving at least 100 tacrolimus in steroid-refractory chronic GVHD. days. The relapse rate in this high-risk, unselected population was only 22%. Overall survival for the entire high-risk population at 1 year is 56% and at 2 years, the corresponding figure is 50%. When stratified by risk, GVHD prophylaxis 61% of low-risk patients remain alive at 2 years, while 43% of high-risk patients remain alive (Figure 3). Since 2000, we have conducted three clinical trials In the HLA-matched, related donor setting, 50 patients evaluating the role of sirolimus as primary prophylaxis have undergone peripheral blood stem cell transplantation against GVHD. In all three studies, sirolimus was given as at the Dana-Farber Cancer Institute using sirolimus and a fixed oral loading dose of 12 mg, 3 days prior to stem cell tacrolimus without methotrexate as GVHD prophylaxis. transplantation and then 4 mg by mouth, per day, in order Data on the first 38 patients surpassing the 100-day mark to maintain a serum trough level of 3–12 ng/ml. are presented in Table 3 (Trial 2). Since sirolimus, In the HLA-matched, unrelated donor and single tacrolimus and methotrexate adequately controlled the antigen-mismatched, related donor setting, over 65 patients rate of GVHD in unrelated donor and mismatched setting, have undergone allogeneic bone marrow transplantation at the hypothesis tested in this trial was that the omission of the Dana-Farber Cancer Institute using the combination methotrexate would not increase the rate of GVHD and of sirolimus, tacrolimus and abbreviated methotrexate would reduce transplant-related toxicity. The median times (5 mg/m2 day þ 1, þ 3, þ 6, þ 11) as GVHD prophylaxis. to neutrophil and platelet engraftment were 14 days (range Updated results on the first 41 patients and survival data 9–17 days) and 13 days (range 10–47 days), respectively. on the entire cohort are presented in Table 3 (Trial 1).23 This is several days shorter than the median times reported Presumably due to a reduction in methotrexate dose, rapid in the literature and is reflective of the omission of neutrophil and platelet engraftment was noted. The methotrexate in this study. The rates of Grades II–IV and incidence of Grades II–IV and III–IV acute GVHD was III–IV acute GVHD were 15 and 4% at 100 days. 26 and 13%, respectively. Overall, the combination was Transplant-related toxicity has been limited: five patients developed severe veno-occlusive disease of the liver (10%) and one patient (2%) developed idiopathic pneumonia Table 3 Summary of clinical trial experience at the Dana-Farber syndrome. CMV reactivation rates were low and no patient Cancer Institute developed an invasive fungal infection during hospitaliza- Trial 1 Trial 2 Trial 3 tion. Mucositis, when assessed prospectively, was moderate and the majority of patients required no parenteral Sample size 41 38 39 nutritional support. Of the 50 patients, 48 survived to first Median age 42 41 57 hospital discharge, which occurred at a median of 19 days HLA match from the infusion of stem cells. Transplant-related mortal- HLA-matched, Related 38 22 ity at 100 days was only 6%. With a median follow-up of HLA-matched, Unrelated 29 17 over 1 year, overall survival at 1 year is 72%24 (Figure 3). 1 Antigen Mismatch 12

Stem cell source Bone marrow || Peripheral blood stem cells |

Conditioning Ablative || Nonmyeloablative |

Methotrexate use || Neutrophil recovery (median days) 18 14 13 (range) (11–32) (9–17) (2–17) Platelet recovery (median days) 29 13 (range) (14–98) (10–47) NA

GVHD (%) Acute Gr. II–IV 26 16 5 Acute Gr. III–IV 13 5 0 Chronic 44 31 48

Survival (%) 100 day treatment-related mortality 15 5 0 Figure 3 Overall survival with sirolimus GVHD prophylaxis and 1 year relapse-free survival 46 69 68 myeloablative conditioning. Group 1: matched, related transplantation; 1 year overall survival 51 72 74 Group 2: unrelated transplantation, favorable risk; Group 3: unrelated transplantation, unfavorable risk.

Bone Marrow Transplantation Sirolimus in allogeneic transplantation C Cutler and JH Antin 474 Using nonmyeloablative conditioning and the combina- transformed lymphocytes.38,39 Sirolimus may be effective in tion of sirolimus, tacrolimus and low-dose methotrexate preventing the growth of lymphocytes infected with other (15 mg/m2 total), 39 patients underwent transplantation members of the herpesvirus family as well. The incidence of from HLA-matched related and unrelated bone marrow reactivation of CMV in our clinical studies was very low transplantation (Table 3, Trial 3). Peritransplant toxicity (o10%). In fact, in our matched, related donor GVHD was expectedly minimal, however, only 5% of patients prophylaxis trial, there was only one case of CMV developed any degree of acute GVHD. Although follow-up reactivation among the first 18 donor–recipient seropositive remains short, the 1 year overall survival estimate is 74%.25 pairs. It is unclear whether sirolimus or the absence of acute Transplantation with sirolimus-based immunosuppres- GVHD and high-dose steroid use was responsible for the sion using alternative donors (haploidentical, single HLA- low rate of viral reactivation. However, it is known that antigen mismatched related and matched unrelated) has CMV specifically upregulates the PI3 kinase/Akt pathway been described as well. Using sirolimus, tacrolimus, during replication and therefore, inhibition of this pathway methotrexate and anti-thymocyte globulin (for haploiden- by sirolimus may be directly responsible for reduced viral tical donors), Claxton et al26 demonstrated stable engraft- reactivation.40,41 Low rates of CMV reactivation have not ment but a 66% rate of acute GVHD. limited to stem cell transplantation, as reports of less frequent CMV reactivation exist for both renal42 and 43 Thrombotic microangiopathy: a potential complication of hepatic transplantation. Similarly, the rates of reactiva- sirolimus use tion of the BK polyoma virus have been noted to be low when sirolimus is used as immunosppression.42 A syndrome of thrombotic microangiopathy, comprised of The broader applicability of sirolimus and the other renal dysfunction, microangiopathic hemolysis and throm- mTOR inhibitors as antineoplastic agents has recently been bocytopenia, occurred in five patients (10%) in our explored. To date, both preclinical and phase I/II clinical matched, related donor study of sirolimus and tacrolimus. trials have been conducted in renal cell carcinoma,44 breast In addition, we have noted the occurrence of thrombotic cancer45,46 and other solid tumors.47–51 It is interesting to microangiopathy in a number of patients receiving note that the side effect profile of these compounds when sirolimus as therapy for established acute GVHD at the used as therapy for solid tumors is strikingly different than Dana-Farber Cancer Institute. the toxicities seen in transplantation. In the randomized, The association of thrombotic microangiopathy with phase II study in renal cancer, the most common toxicities cyclosporine or tacrolimus after allogeneic stem cell included maculopapular rash (76%), mucositis (70%), transplantation and solid-organ transplantation is well asthenia (50%) and nausea (43%).44 known.27,28 Recently, the combination of sirolimus and In addition, the activity of mTOR inhibitors has been calcineurin inhibitors has been associated with the syn- tested in hematologic neoplasms that are currently treated drome.29,30 Sirolimus may promote thrombotic microan- by allogeneic stem cell transplantation.52 Brown et al53 giopathy via direct endothelial damage or may potentiate demonstrated suppression of lymphoblastic leukemia the effects of calcineurin inhibitors;31,32 however, there is growth both in vitro and in a transgenic mouse model only one case report of de novo thrombotic microangio- modulated by sirolimus’ inhibition of the p70 S6 kinase pathy related to sirolimus monotherapy.33 The mechanism pathway. Similarly, Decker et al54 have demonstrated of sirolimus-induced microangiopathy may be molecular similar findings using CLL as a model. mTOR inhibitors mimicry between sirolimus and tacrolimus; however, are also being tested as a rationale target in CML, since the conversion from tacrolimus to sirolimus as therapy for BCR-ABL kinase promotes cell cycling through the p70 S6 thrombotic microangiopathy has been reported without kinase and the PI3 kinase pathway.55,56 Other future apparent untoward effects.34–36 In all cases reviewed at the applications of sirolimus and other mTOR inhibitors are Dana-Farber, thrombotic microangiopathy resolved spon- displayed in Table 4. taneously when tacrolimus was discontinued and sirolimus Sirolimus, when used as part of combination immuno- was held, in the event of supratherapeutic levels. No long- suppression after allogeneic stem cell transplantation is safe term renal complications have been noted, to date. We strongly recommend that when used in combination with a calcineurin agent, levels of both sirolimus and the Table 4 Future applications of sirolimus and other mTOR calcineurin inhibitor are monitored frequently. inhibitors Application Rationale

GVHD prophylaxis Evidence of efﬁcacy in three Future applications of sirolimus and mTOR inhibitors: clinical trials harnessing antiviral and antineoplastic properties in Treatment of established acute Prophylaxis effectiveness and transplantation and chronic GVHD anecdotes suggest efﬁcacy Prevention of viral reactivation Low rates of viral reactivation Sirolimus has been used successfully in conjunction with after transplantation after transplantation and ability to suppress viral growth in vitro via Rituximabas therapy for post transplant lymphoprolifera- mTOR inhibition tive disorder in three cases.37 Supporting the reported Treatment of post transplant Ability to suppress in vitro growth favorable clinical results is the fact that in vitro, everolimus lymphoproliferative disorder of EBV-transformed cell lines (Certicans, Novartis), a sirolimus analog, as well as Treatment of hematological mTOR interference disrupts many malignancies and solid tumors pathways involved in neoplasia sirolimus are capable of suppressing the growth of EBV-

Bone Marrow Transplantation Sirolimus in allogeneic transplantation C Cutler and JH Antin 475 and highly effective in reducing the incidence and severity 12 Fairbanks KD, Eustace JA, Fine D, Thuluvath PJ. Renal of acute GVHD. Fears that more efficient GVHD function improves in liver transplant recipients when switched prophylaxis may lead to less potent GVL responses may from a calcineurin inhibitor to sirolimus. Liver Transpl 2003; 9: be unfounded if the GVHD prophylaxis agent itself has 1079–1085. direct antitumor activity. If in addition, this GVHD 13 Nair S, Eason J, Loss G. Sirolimus monotherapy in prophylaxis agent were capable of controlling viral infec- nephrotoxicity due to calcineurin inhibitors in liver transplant recipients. Liver Transpl 2003; 9: 126–129. tions after transplantation, then this agent would certainly 14 Cotterell AH, Fisher RA, King AL et al. Calcineurin inhibitor- become an important part of all GVHD prophylaxis induced chronic nephrotoxicity in liver transplant patients is regimens. Randomized trials to confirm these preliminary reversible using rapamycin as the primary immunosuppressive findings and hypotheses are in development at this time. agent. Clin Transplant 2002; 16 (Suppl. 7): 49–51. 15 McAlister VC, Gao Z, Peltekian K et al. Sirolimus–tacrolimus combination immunosuppression. Lancet 2000; 355: 376–377. 16 Koenen H, Michielsen E, Verstappen J et al. Superior T-cell Acknowledgements suppression by rapamycin and FK506 over rapamycin and cyclosporine A because of abrogated cytotoxic T-lymphocyte This study was supported in part by P01 HL070149 from the induction, impaired memory responses, and persistent apop- National Heart, Lung, and Blood Institute and by an unrest- tosis. Transplantation 2003; 75: 1581–1590. ricted educational grant from Fujisawa Healthcare, Inc. 17 Ciancio G, Burke GW, Gaynor JJ et al. 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