Heart transplantation (HTx) is still the only complete

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1 Everolimus-Incorporated Immunosuppressant Strategy Improves Renal Dysfunction While Maintaining Low Rejection Rates fter Heart Transplantation in Japanese Patients Teruhiko Imamura, 1 MD, Koichiro Kinugawa, 2 MD, Minoru Ono, 3 MD, Yukie Kagami, 4 RN, Miyoko Endo, 4 RN, Shun Minatsuki, 1 MD, Hironori Muraoka, 1 MD, Naoko Kato, 1 PhD, Toshiro Inaba, 1 MD, Hisataka Maki, 1 MD, Masaru Hatano, 1 MD, tsushi Yao, 1 MD, Shunei Kyo, 2 MD, and Issei Komuro, 1 MD Summary The long-term survival of heart transplantation (HTx) recipients has increased significantly in recent years, however, the nephrotoxic adverse effects of calcineurin inhibitors (CNIs) are still a major concern. Recently, an inhibitor of mammalian target of rapamycin, everolimus (EVL), has emerged as an alternative immunosuppressant drug that may allow CNI dosage reduction and thereby spare renal function. Data were collected from 20 HTx recipients who had received EVL (target trough level 3-8 ng/ml) along with a dose reduction of CNIs and/or mycophenolate mophetil (MMF) and had been followed for 1 year. Estimated glomerular filtration rate increased significantly with a reduction in the CNI dosage in a dose-dependent manner (P < 0.001, r = ). Neutrophil count increased significantly (P < 0.05) with a reduction in the dosage of MMF (P = 0.009, r = ). Cytomegalovirus antigenemia remained negative after EVL administration among all candidates without any antiviral agents (P = 0.001). There were no significant increases in the acute rejection rates among recipients with EVL compared to those without EVL (P = 0.132). n immunosuppressant strategy incorporating EVL could reduce the CNI and MMF dosages, which resulted in improvements in renal dysfunction and neutropenia while maintaining low rejection rates among HTx recipients. (Int Heart J 2013; 54: ) Key words: Renal function, Mycophenolate mophetil, Calcineurin inhibitor Heart transplantation (HTx) is still the only complete solution for stage D heart failure 1,2) owing to not only recent improvements in surgical procedures but also to newly developed immunosuppressant therapy. s one of the essential immunosuppressant drugs, calcineurin inhibitors (CNIs) have been widely used after HTx for 30 years around the world. 3) There is no doubt that this revolutionary class of drugs led to the current marked improvement in survival rates, however, the adverse effects of CNI therapy, especially the worsening of renal function, remain a concern. 4) The inosine monophosphate dehydrogenase inhibitor mycophenolate mophetil (MMF) has become one of the standard immunosuppressant regimens for HTx maintenance therapy, 5) however, MMF is often associated with serious neutropenia. 6) Therefore, a remaining major challenge is to develop immunosuppressive strategies that allow CNI/MMF minimization or elimination in order to reduce adverse events while maintaining low acute rejection rates. Recently, a new immunosuppressive drug, everolimus (EVL), which is an inhibitor of mammalian target of rapamycin, has been approved for HTx recipients in Europe and Japan. 7) EVL binds to the tacrolimus-binding protein and creates an active moiety that blocks the mammalian target of rapamycin and then arrests the cell cycle of lymphocytes in the G1 phase. 8) EVL exerts immunosuppressive effects by inhibition of interleukin-2-mediated lymphocyte proliferation. 8) Recent studies have suggested that the administration of EVL along with reduction or elimination of CNIs could spare renal function in heart or kidney transplantation recipients. 9-18) However, to the best of our knowledge, the relationship between the dosage of a standard immunosuppressant drug and the abovementioned adverse effects has not yet been fully analyzed, especially in Japanese patients. In the present study, we analyzed the dose-dependent adverse effects of EVL, CNIs, and MMF. From the Departments of 1 Cardiovascular Medicine, 2 Therapeutic Strategy for Heart Failure, 3 Cardiothoracic Surgery, and 4 Organ Transplantation, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. This work was supported in part by the FUGKU Trust for Medicinal Research to K.K., grants for clinical epidemiology research from the Japanese Heart Foundation, the Japanese ssociation for CerebroCardiovascular Disease Control, and strazeneca to N.K. and K.K, a domestic collaborative research grant from the Pfizer Health Research Foundation to N.K. and K.K., and a Grant-in-id for a JSPS Postdoctoral Research Fellow from the Japan Society for the Promotion of Science (no ) to N.K. ddress for correspondence: Koichiro Kinugawa, MD, Department of Therapeutic Strategy for Heart Failure, Graduate School of Medicine, The University of Tokyo, Hongo, unkyo-ku, Tokyo , Japan. Received for publication February 13, Revised and accepted March 10,

2 Vol 54 No 4 EVEROLIMUS WITH REDUCED CNI 223 Methods Patient population: The study patients included 25 consecutive recipients who had undergone HTx at the University of Tokyo Hospital and 11 consecutive patients who received a HTx in a foreign country. They were followed at the University of Tokyo Hospital between June 2006 and December EVL was administered to 22 (61.1%) patients. Two patients were excluded because of insufficient data. The final patient population was 20 patients. ll patients were followed for at least 1 month, and 14 patients for 1 year. The institutional protocol for the monitoring of rejection with serial endomyocardial biopsy and hemodynamic measurements routinely began 7 days after transplantation and were then performed weekly in the first month, every other week until 3 months, monthly until 6 months, and then once a year thereafter. When rejection was suspected, these examinations were performed on an emergency basis. Written informed consent was obtained from all patients and/or family members before a HTx. The study protocol was approved by the Ethics Committee of the Graduate School of Medicine, the University of Tokyo [application number 779 (1)]. Immunosuppressant strategy: fter HTx, all recipients were treated with a standard immunosuppressive regimen consisting of a CNI [cyclosporine (Cy) or tacrolimus (TC)], MMF, and low dose prednisolone. The trough concentration of Cy was maintained at ng/ml until 3 months, ng/ ml until 6 months, ng/ml until 1 year, and ng/ml after 1 year. The trough concentration of TC was maintained at ng/ml until 3 months after HTx, around 10 ng/ml until 1 year, and 5-8 ng/ml after 1 year. MMF was administered at a dose of mg daily during the study period unless an adverse effect occurred. Prednisolone was administered at 1 mg/kg daily at first, and then tapered off gradually until 1 year after HTx if possible. The timing of the introduction of EVL was determined by the attending physician based on the following criteria: (1) Conversion from MMF to EVL because of neutropenia or digestive symptoms such as repeated diarrhea. (2) dministration of EVL with reduction of CNI because of deteriorating renal function (a continuous rise in serum creatinine level over 1.5 mg/dl). In this case, the target CNI trough concentration was reduced by approximately 30%. (3) dd-on treatment of EVL because of progressive cardiac allograft vasculopathy (progression of maximal intimal thickness > 1.5 mm/year on intravascular ultrasound) or frequent acute cellular rejection (ISHLT grade 2R). ll introductions of EVL except for one case were initiated in-hospital and the recipients were carefully monitored until the trough concentration of the immunosuppressant had stabilized. ll patients achieved the target EVL trough concentration range (3-8 ng/ml) throughout most of the study period. 19) Variables evaluated: t baseline (ie, at the time of EVL introduction) and 1 month and 1 year after the administration of EVL, a physical examination was performed, laboratory data were assayed, and the trough concentrations of CNIs and EVL were monitored. t each routine admission or unexpected admission due to suspected rejection, hemodynamic study and endomyocardial biopsy were performed. Seventeen recipients who did not receive EVL were also analyzed as a control group. Statistical analysis: ll statistical analyses were performed using statistical software (SPSS Inc, Chicago, IL, US). Results are presented as the mean ± standard deviation unless otherwise specified. Two-sided P < 0.05 was considered statistically significant. Demographic, laboratory, and echocardiographic parameters at 1 month and 1 year after the administration of EVL were compared with those of baseline using Dunnett s post hoc test. Correlations were determined using Pearson s correlation coefficient. Results Patient baseline characteristics (Table): The study population included 15 males (75.0%) with a mean age of 41.6 ± 13.1 years (range, years-old). total of 20 recipients were enrolled and followed for 1 month and 14 of these 20 were followed for 1 year after the administration of EVL. No patient discontinued EVL during the study period. Eight patients (40.0%) had received Cy, and its trough concentration was reduced significantly after the administration of EVL (P < 0.05). Twelve patients (60.0%) had received TC, and its trough concentration was reduced significantly after the administration of EVL (P < 0.01). CNI administration was not discontinued in any patient during the study period. ll patients had received MMF at baseline. Twelve patients discontinued MMF by the first month after the administration of EVL, and one discontinued MMF by the first year after the administration of EVL. No patients restarted MMF during the study period. The main indication for the administration of EVL was (1) a combination of CNI-induced renal impairment and MMF-induced neutropenia [6 patients (30.0%)], (2) MMF-induced neutropenia and diarrhea [6 patients (30.0%)], (3) progression of CV [6 patients (30.0%)], and (4) recurrent rejection episodes that developed against triple combination therapy including CNI, MMF, and prednisolone [2 patients (10.0%)]. The neutrophil count averaged 2409 ± 1846/μL at baseline, and increased significantly after the administration of EVL (P < 0.01). Mean egfr was 58.9 ± 33.6 ml/minute/1.73 m 2 at baseline, increased to 71.3 ± 39.8 ml/minute/1.73 m 2 after 1 month (P = versus baseline), and stabilized at 76.1 ± 49.4 ml/minute/m 2 after 1 year (P < versus baseline). ll lipid parameters including total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides had increased slightly after 1 month but returned to baseline after 1 year. Cytomegalovirus (CMV) antigenemia was positive in 12 recipients (60.0%) at baseline, and CMV antigenemia assay remained negative in all recipients after the administration of EVL without any antiviral drug administration. Immunosuppressant and renal function: Relative changes in the trough concentrations of CNIs showed a strong correlation with %changes in estimated glomerular filtration rate (egfr) after the administration of EVL (r = and P = after 1 month, r = and P < after 1 year) (Figure 1). The trough concentration of EVL was slightly negatively correlated with %changes in egfr at 1 month after the administration of EVL (r = , P = 0.058) (Figure 2). There was no correlation with the changes in dose of MMF and %changes in egfr at 1 month after the administration of EVL (r = 0.269, P

3 224 IMMUR, ET L Int Heart J July 2013 Table. Data at aseline, fter 1 Month, and 1 Year fter the dministration of Everolimus aseline (n = 20) fter 1 month (n = 20) P fter 1 year (n = 14) P Demographic parameters ge, years 41.6 ± ± ± Male, n (%) 15 (75.0) 15 (75.0) - 10 (76.9) - ody height, cm ± ± ± ody weight, kg 57.0 ± ± ± ody mass index 1.62 ± ± ± Immunosuppression drug Co of everolimus, ng/ml 0 (n = 20) 5.5 ± 1.7 (n = 20) < * 5.6 ± 1.9 (n = 14) < * Co of cyclosporine, ng/ml ± 46.6 (n = 8) ± 59.2 (n = 8) * ± 63.9 (n = 5) * Co of tacrolimus, ng/ml 10.8 ± 2.9 (n = 12) 7.0 ± 1.2 (n = 12) < * 7.6 ± 1.5 (n = 9) Dose of MMF, mg/day 1525 ± 638 (n = 20) 638 ± 992 (n = 20) < * 672 ± 1036 (n = 14) < * Laboratory data Neutrophil count, /μl 2201 ± ± 1484 < * 3357 ± * Serum sodium, meq/l ± ± ± Serum potassium, meq/l 4.4 ± ± ± Serum blood urea nitrogen, mg/dl 21.4 ± ± ± egfr, ml/minute/m ± ± * 76.1 ± 49.4 < * Serum total albumin, g/dl 4.1 ± ± ± Serum total bilirubin, mg/dl 0.7 ± ± ± Plasma NP, log 10 pg/ml 1.94 ± ± ± Total cholesterol, mg/dl ± ± ± LDL cholesterol, mg/dl ± ± ± HDL cholesterol, mg/dl 60.9 ± ± ± Triglycerides, mg/dl ± ± ± Cytomegalovirus antigenemia, n (%) 12 (60.0) Echocardiographic parameters LV diastolic diameter, mm 43.1 ± ± ± Ejection fraction, % 68.5 ± ± ± Co indicates trough concentration; MMF, mycophenolate mophetil; egfr, estimated glomerular filtration ratio; NP, -type natriuretic peptide; LDL, low-density lipoprotein; HLD, high-density lipoprotein; and LV, left ventricle. * P < 0.05 compared with that of baseline using Dunnett s post hoc test. P < versus baseline by chi-square test with Tukey s test. Figure 1. Relationship between %changes from baseline in trough concentration of calcineurin inhibitors and %changes from baseline in egfr at 1 month () and 1 year () after the administration of EVL. egfr indicates estimated glomerular filtration rate; EVL, everolimus; TC, tacrolimus; Cy, cyclosporine; and CNI, calcineurin inhibitor. Figure 2. Relationship between %changes from baseline in egfr at 1 month after the administration of EVL and trough concentration of EVL () and relationship between %changes from baseline in egfr at 1 month after the administration of EVL and changes in dose of MMF (). egfr indicates estimated glomerular filtration rate; EVL, everolimus; and MMF, mycophenolate mophetil. = 0.252) (Figure 2). Figure 3 shows that baseline egfr was slightly negatively correlated with the %changes in egfr among patients with mild to severely progressed renal dysfunction (< 90 ml/minute/1.73 m 2 of egfr) at 1 month after the administration of EVL (r = , P = 0.174). There was no correlation between the timing of EVL administration and the improvement of renal function at 1 month after the administration of EVL (r = 0.264, P = 0.298) (Figure 3). Immunosuppressant and neutrophil count: ll recipients had received MMF before the administration of EVL, and the mean dose was 1525 ± 638 mg/day. The mean dose of MMF after 1 month and 1 year was 638 ± 992 and 672 ± 1036 mg/

4 Vol 54 No 4 EVEROLIMUS WITH REDUCED CNI 225 Figure 3. Relationship between %changes from baseline in egfr at 1 month after the administration of EVL and baseline egfr () and timing of the administration of EVL (). egfr indicates estimated glomerular filtration rate; EVL, everolimus; and HTx, heart transplantation. Figure 4. Relationship between changes in dose of MMF and %changes from baseline in neutrophil count at 1 month () and 1 year () after the administration of EVL. EVL indicates everolimus and MMF, mycophenolate mophetil. day, respectively, values that were significantly reduced compared with baseline (P < 0.001). Changes in MMF dosage had a strong negative correlation with the %changes in neutrophil count after the administration of EVL (r = and P = after 1 month, r = and P = after 1 year) (Figure 4). There was no correlation between the %changes in neutrophil count and trough levels of EVL or the %changes in the trough concentrations of CNIs (r = 0.219, P = 0.213, and r = 0.184, P = 0.184, respectively). dverse events: There were no major adverse events associated with EVL such as thrombocytopenia, anemia, lymphocoele, pneumonitis, impaired wound healing, or proteinuria, although stomatitis was common in almost every patient treated with EVL. Two patients experienced rejection; ISHLT grade 2R on day 623 and grade 3R at day 30, after the administration of EVL, and each rejection was treated by single steroid pulse therapy. Kaplan-Meier analysis showed that the proportion of patients free from rejection among HTx recipients with EVL treatment was slightly better than those without EVL (P = 0.132) (Figure 5). Figure 5. Proportion of patients free from rejection after HTx among patients with/without EVL. EVL indicates everolimus and HTx, heart transplantation. Discussion In the present study, we administered EVL, an inhibitor of mammalian target of rapamycin, along with a reduction or discontinuation of MMF and/or CNIs in HTx recipients and then followed them for 1 year. We found that the %increase in egfr or neutrophil count was significantly correlated with the %decrease in the dosage of CNI or MMF, respectively. Moreover, we could introduce EVL safely while maintaining rejection rates as low as a standard immunosuppressant regimen without EVL. Recently, EVL has been demonstrated to be advantageous in various aspects, including prophylactic potentials of CMV infection and cardiac allograft vasculopathy (CV), 20-24) a less myelosuppressive character compared with MMF, 20) and less renal toxicity compared with CNIs. 9,11,15-17,25,26) Several recommendations for an indication for EVL have been recently proposed considering the above advantages of EVL, 12-14) and we initiate EVL administration in principle on the basis of these recommendations as mentioned in the methods. With respect to CMV infection, we previously reported that EVL might inhibit CMV proliferation through down-regulation of the phosphatidylinositol 3-kinase pathway, whose activation is essential for viral replication, 20,21) Consistently with previous clinical reports, we found no patients had a CMV infection after the introduction of EVL even though several recipients were CMV-seronegative. It has been suggested that CMV infection plays an important role in the pathogenesis of CV, 27) and minimizing CMV infections may be one of the key mechanisms by which EVL prevents CV. 24) MMF is one of the standard immunosuppressants for HTx recipients, and neutropenia is not uncommonly associated with MMF treatment. 26) Neutropenia is further worsened when MMF is administered simultaneously with valganciclovir for the treatment of CMV infection because of their synergistic myelosuppressive effects as we also previously reported. 20,28) lthough there are few reports on the dose-dependent effects

5 226 IMMUR, ET L Int Heart J July 2013 of MMF in neutropenia, we have demonstrated that %increases in neutrophil count were closely correlated with %decreases in the dose of MMF at 1 month after the administration of EVL, and that the trend continued for 1 year after the administration of EVL. Digestive tract symptoms including diarrhea are also notorious adverse effects of MMF, 26) and avoiding diarrhea is desirable in order to ensure absorption of immunosuppressant drugs through the gastrointestinal tract. fter switching to EVL from MMF, no patient in the present study suffered digestive tract symptoms. These results indicate, therefore, that EVL can be a real alternative to MMF. Recent studies have demonstrated that administration of EVL along with reduced CNI administration could spare renal function among patients who underwent heart or kidney transplantation. 9,11,26) n expert panel reported that the introduction threshold should be a serum creatinine concentration of 2.0 mg/dl. 15) We normally commence EVL administration if serum creatinine levels exceed 1.5 mg/dlwhich takes into consideration a different calculation method for egfr in Japanese patients. mong recipients with an egfr < 90 ml/ minute/1.73 m 2, we found that CNI reduction met much more increases in egfr in the patients with lower baseline renal function. This observation indicates that patients with decreased renal function might receive more benefit from CNI reduction. However, it is believed that recipients with advanced renal dysfunction (egfr < 30 ml/minute/1.73 m 2 ) would not respond to CNI reduction because of depletion of their recovery potential. In fact, the German-ustrian Certican Consensus Conference recommended that recipients should have a GFR of > 40 ml/minute/1.73 m 2 to benefit substantially from a reduction of CNI while adding EVL considering their potential for recovery of renal function. 12) We could not address this apparent discrepancy, since there were no patients with such advanced renal dysfunction (< 30 ml/minute/1.73 m 2 of egfr) in this study. Only a few reports have analyzed the quantitative relationship between the amount of CNI reduction and recovery of renal function. 29) We demonstrated here quantitatively that %decreases in the dose reduction of CNI were closely correlated with %increases in egfr at 1 month after the administration of EVL, and that the trend continued for 1 year after the administration of EVL. How much should the dose of CNI be reduced when EVL will be administered? The relative reductions of Cy and TC in our study were 36.8% and 36.4%, respectively, and were similar to previous recommendations (25-50% dose reduction of CNI) ) It was reported that complete withdrawal of a CNI after EVL initiation could further improve renal function without risk of rejection after heart or kidney transplantation. 16,17) However, a CNI-free immunosuppressant regimen with EVL has not yet replaced a conventional CNI-dominant regimen because of a lack of evidence. In fact, a CNI-free regimen had a slightly higher probability of rejection 18) and might increase donor-specific HL antibodies among renal transplantation recipients. 30) second issue is when EVL should be initiated after HTx. Gude, et al recently reported that an improvement in renal function after switching to EVL treatment was more likely within the first year post-htx (early group) compared with several years after HTx (late group), because a long-injured kidney might have lost the ability to recover even after the discontinuation of CNI. 10) Inconsistently with their reports, we found no correlation between the timing of EVL initiation and amount of recovery in renal function, which was probably because EVL was started within 1 year after HTx in most of our patients. However, in our study, a couple of patients in whom EVL was initiated several years after HTx also benefitted from CNI reduction in terms of renal function. Further studies are needed to clarify this point. n association between EVL and impaired wound healing after major surgery due to the inhibition of fibroblast proliferation by EVL has been reported. 31) Wound healing is an important issue, especially in Japanese HTx recipients since approximately 90% of them receive ventricular assist devices during their waiting period. 32) The median duration until EVL introduction was 349 days post-htx in our institute, but 2 patients were administered EVL within 2 months after bridging to HTx from ventricular assist devices without any complications, including incomplete wound healing. In fact, several groups have recommended that EVL could be started even as little as 1 week after normal surgery ) The risk of CMV infection is extremely high among CMV-seronegative recipients given a CMV-seropositive donor heart. 33) Introduction of EVL with reduced CNI as early as 2 months after HTx might be considered in such a specific situation, with an expectation it will have a prophylactic effect on CMV infection ) EVL has been reported to be associated with adverse effects that include renal toxicity and a worsening of lipid metabolism. Several authors reported that EVL inhibits glomerular endothelial cell proliferation and vascular endothelial growth factor, and eventually may decrease renal function, albeit to a relatively modest degree. 34,35) We consistently found that higher trough levels of EVL had a weak but negative impact on egfr. lthough the renal toxicity of EVL is close to negligible compared with that of CNI, longer administration of EVL may have the disadvantage to preserve renal function. Evidence based on further observation is still needed. With regards to lipid metabolism, EVL has been reported to worsen hypercholesterolemia and hypertriglyceridemia by inhibiting the degradation and excretion of cholesterol. 36,37) Though the serum concentrations of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides were slightly increased 1 month after the administration of EVL, these lipid parameters had returned to baseline after 1 year by titration of statin or additional administration of ezetimibe. The adverse effects of EVL on lipid metabolism may be managed sufficiently in the current era in which potent antihyperlipidemic drugs are readily available. 5,13) The present study has several limitations. First, it was conducted in a single center, and consequently included a limited number of patients because of a shortage of donor hearts in Japan. We expect the number of HTx to increase due to amendment of the organ transplantation law in July 2010, which will strengthen our analyses. Second, there was a limitation associated with patient selection bias, and further investigations in prospective trials should be performed. Third, we analyzed the effect of CNI reduction with administration of EVL for 1 year after HTx. longer observation period would be needed to analyze the long-term efficacy, safety, and tolerability of EVL. In conclusion, an immunosuppressant regimen consisting of dose reductions of CNIs and MMF together with the initiation of EVL administration can overcome drug-induced ad-

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