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Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients
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Clinics and Research in Hepatology and Gastroenterology (2019) xxx, xxx—xxx
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ORIGINAL ARTICLE
Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients M. Neuberger a,1, C. Sommerer b,1, S. Böhnisch b, N. Metzendorf b, A. Mehrabi c, W. Stremmel a, D. Gotthardt a, M. Zeier b, K.H. Weiss a, C. Rupp a,∗ a
University Hospital Heidelberg, Internal Medicine IV, 69120 Heidelberg, Germany University Hospital Heidelberg, Division of Nephrology, 69120 Heidelberg, Germany c University of Heidelberg, Department of General, Visceral, and Transplantation Surgery, 69120 Heidelberg, Germany b
KEYWORDS Liver transplantation; Immunosuppression; IMPDH; Mycophenolic acid; Limited sampling strategy
∗ 1
Summary Background: Due to the development of immunosuppressants, the focus in transplanted patients has shifted from short-term to long-term survival as well as a better adjustment of these drugs in order to prevent over- and under-immunosuppression. Mycophenolic acid (MPA) is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) and approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation, where it has become a part of the standard therapy. Targeting inosine monophosphate IMPDH activity as a surrogate pharmacodynamic marker of MPA-induced immunosuppression may allow a more accurate assessment of efficacy and aid in limiting toxicity in liver transplanted patients. Aim: Assess IMPDH-inhibition in liver transplant recipients and its impact on biliary/infectious complications, acute cellular rejection (ACR) and liver dependent survival. Methods: This observational cohort study comprises 117 liver transplanted patients that were treated with mycophenolate mofetil (MMF) for at least 3 months. Blood samples (BS) were collected and MPA serum level and IMPDH activity were measured before (t(0)), 30 minutes (t(30)) and 2 h after (t(120)) MMF morning dose administration. Regarding MPA, we assessed the area under the curve (AUC). Patients were prospectively followed up for one year and assessed for infectious and biliary complications, episodes of ACR and liver dependent survival. Results: The MPA levels showed a broad interindividual variability at t(0) (2.0 ± 1.8 ng/ml), t(30) (12.7 ± 9.0 ng/ml) and t(120) (7.5 ± 4.3 ng/ml). Corresponding IMPDH activity was at t(o) (23.2 ± 9.5 nmol/h/mg), at t(30) (16.3 ± 8.8 nmol/h/mg) and t(120) (18.2 ± 8.7 nmol/h/mg). With regard to MPA level we found no correlation with infectious or biliary complications
Corresponding author. University Hospital Heidelberg, Department of Gastroenterology, INF 410, 69121 Heidelberg, Germany. E-mail addresses: [email protected], [email protected] (C. Rupp). Both authors contributed equally.
https://doi.org/10.1016/j.clinre.2019.12.001 2210-7401/© 2019 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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M. Neuberger et al. within the follow-up period. Patients with baseline IMPDH(a) below the median had significant more viral infections (6 (10.2%) vs. 17 (29.3%); P = 0.009) with especially more cytomegalovirus (CMV) infections (1 (3.4%) vs. 6 (21.4%); P = 0.03)). Furthermore, patients with baseline IMPDH(a) above the median developed more often non-anastomotic biliary strictures (8 (13.6%) vs. 1 (1.7%), P = 0.03). We found the group reaching the combined clinical endpoint of death and re-transplantation showing significantly lower MPA baseline values (t(0) 0.9 ± 0.7 vs. 2.1 ± 1.8 g/ml Mann-Whitney-U: P = 0.02). We calculated a simplified MPA(AUC) with the MPA level at baseline, 30 and 120 minutes after MPA administration. Whereas we found no differences with regard to baseline characteristics at entry into the study patients with MPA (AUC) below the median experienced significantly more often the combined clinical endpoint (12.1% (7/58) vs. 0.0% (0/57); P = 0.002) and had a reduced actuarial re-transplantation-free survival (1.0 year vs. 0.58 years; Log-rank: P = 0.007) during the prospective one-year followup period. In univariate and multivariate analysis including gender, age, BMI, ACR, MPA (AUC) and IMPDH(a) only BMI, MPA (AUC) and IMPDH(a) were independently associated with reduced actuarial re-transplantation-free survival. Conclusion: MPA-levels and IMPDH-activity in liver transplanted patients allows individual risk assessment. Patients with higher IMPDH inhibition acquire more often viral infections. Insufficient IMPDH inhibition is associated with development of non-anastomotic bile duct strictures and reduced re-transplantation-free survival. © 2019 Elsevier Masson SAS. All rights reserved.
Introduction Due to the development of immunosuppressants the focus in transplanted patients has shifted from short-term to longterm survival as well as a better adjustment of these drugs in order to prevent over- and under-immunosuppression [1,2]. Mycophenolic acid (MPA) is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) and approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation, where it has become a part of the standard therapy [3—6]. Targeting inosine monophosphate IMPDH activity as a surrogate pharmacodynamic marker of MPA-induced immunosuppression may allow a more accurate assessment of efficacy and aid in limiting toxicity in liver transplanted patients [7—9]. We aimed to assess the effect of IMPDH inhibition in liver transplant recipients and evaluate the impact on biliary and infectious complications, episodes of acute cellular rejections (ACR) and liver dependent survival.
Patients and methods
Table 1
Baseline characteristics.
Patients Gender Male Female Age at serum sampling Age at liver transplantation Immunsuppression MMF+ Cyclosporine Tacrolimus Further characteristics BMI [kg/m2 ] Serum bilirubine [mg/dl] Serum creatinine [mg/dl] INR MELD Leucocytes [/nl] Lymphocytes [%] Thrombocytes [/nl] GOT [U/l] GPT [U/l]
117 94 (80.3%) 23 (19.7%) 561 ± 10.8 50.8 ± 12.0 53 (45.3%) 64 (54.7%) 26.8 ± 5.16 1.06 ± 2.08 1.22 ± 0.95 1.05 ± 0.25 9.2 ± 4.03 5.9 ± 2.05 22.6 ± 7.96 186.7 ±74.17 30.4 ± 27.21 32.41 ± 42.01
Study design In this prospective cohort study blood samples (BS) were withdrawn and MPA serum levels and IMPDH activity were measured in 117 stable liver transplant patients. BS were collected from January throughout June 2014 during the patients’ post-transplant follow-up appointments in our outpatient clinic. Therefore, all patients having an appointment in our outpatient department during the study period were screened for MMF + CNI and included consecutively. No patient was excluded for any other reason than a
differing immunosuppressant regimen. ‘‘Stable’’ was defined as being seen in an outpatient setting, not being on steroids or showing clinical or laboratory signs of infection or hepatic decompensation and having a stable CNI dosing resulting in the specified CNI target range. This is mirroring in an average MELD score of 9.2 ± 4.0 and further laboratory markers for infection, kidney and liver function within the normal range (see Table 1). Median time from transplantation was 4.7 ± 5.2 years. Patients admitted to our ward, showing signs of acute rejection
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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Effect of mycophenolic acid in liver transplant patients or on prolonged steroid therapy were excluded as well as patients with proven CMV infection. The study patients have already been on MMF for at least 3 months. Blood samples were taken before (t(0)), 30 minutes (t(30)) and 2 hours after (t(120)) MMF morning dose administration. The MMF dosing was 2000 mg/day (87,2%) in vast majority of patients included into the study. This reflects the standard dose rate of MMF in combined IS regimen in stable patients. The residual patients were on 1000 mg/day (10,3%) or had individual MMF dosing (500 mg—3000 mg per day) adjusted to their needs, which were figured out and approached to in post-transplant follow-up, especially balanced between an impaired kidney-function and adverse drug reactions. All patients were on double immunosuppression (IS) consisting of MMF and either cyclosporin A or tacrolimus. Patients were followed up prospectively for one year, starting at the time of sampling, and assessed for infectious and biliary complications, episodes of ACR and liver dependent survival. All ACRs were biopsy-proven and reviewed by an experienced gastrointestinal pathologist, whereas the anastomotic and non-anastomotic biliary strictures were diagnosed endoscopically. The follow-up period was 11 to 16 months. We assessed information in the post-transplant follow-up care clinic, where patients were seen every 3, 6 or 12 months depending on the interval to transplantation and their clinical course. Urgent complications were registered there as well since the post-transplant follow-up care clinic is the first point of contact for this highly selective patient population. Additionally, we assessed the patient’s clinical course by screening the routine follow-up reports as well as the urgent complications in the corresponding electronic patients’ records.
MPA & IMPDH measurement The measurement of MPA levels has been performed by the routine laboratory of the University Hospital Heidelberg using the EMIT (enzyme-multiplied immunoassay technique (EMIT; Dimension XL, Dade Behring, Marburg, Germany)). The isolation and incubation process of peripheric blood monocytic cells (PBMC) and the determination of the IMPDH activity using high performance liquid chromatography (HPLC) have been performed in the laboratories of our working groups, where this method has been used and published before to determine the IMPDH activity in renal transplant patients [10]. As a first step the PBMCs have to be isolated from lithiumheparin whole blood sample. The method we used has initially been established by Böyum et al. in 1968 [11] and was modified and improved by Glander et al. [12,13]: The lithium-heparin whole blood sample was mixed with phosphate buffered saline (PBS) and stacked as a layer on top of Ficoll. After centrifugation that mixture for 20 minutes (room temperature, 2500 rpm) the PBMCs presented in a separate layer and could be taken to another sample tube with the help of a pipette. During this next step the PBMCs were washed with 5 ml of PBS and centrifugated again (room temperature, 2500 rpm). In this way PBMC pellets formed, which were brought into solution with 250 l of HPLC water. These cell lysates were stored at -80 ◦ C.
3 In order to determine the IMPDH-activity next to a chromatographic and a radiometric method there is a method available using real-time polymerase chain reaction (PCR) [14]. The determination of IMPDH activity using HPLC seems the best accessible and most widely used one [15]. Compared to other methods HPLC reduces the variability of results and provides quick results [14,15]. This method’s basis is the reaction of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), which is catalyzed by IMPDH. After incubating the PBMCs with IMP and NAD+ we could quantify XMP using HPLC with subsequent ultraviolet (UV) detection. After determining the protein concentration and knowing the amount of XMP we could calculate the IMPDH activity in ‘‘nmol/h/mg Protein’’. For protein determination we used Thermo Scientific’s ‘‘Pierce BCA Protein Assay Kit (#23225)’’. For IMPDH the values were assessed separately whereas regarding MPA the area under the curve (AUC(0—2 h)) was calculated using a limited sampling strategy (LSS) [10].
Ethical approval Blood withdrawal, data collection and prospective follow-up were performed with approval of the local ethics committee (‘‘Ethikkommission Medizinische Fakultät Heidelberg’’, S-043/2011). Written informed consent was obtained from all volunteers.
Statistical analysis Calculations were carried out using IBM® SPSS® Statistics 20 for Windows (SPSS Inc., Chicago, IL, USA)). Frequencies were compared using the chi-squared test or the Fisher’s exact test, where appropriate. Continuous data were compared using the nonparametric Wilcoxon rank-sum test. Actuarial transplantation-free survival was estimated using a Kaplan-Meier product limit estimator. Differences between the actuarial estimates were tested using the log rank test. Factors that independently affected the risk of reduced transplantation-free survival were determined using Cox proportional hazard ratio models with simultaneous adjustment for gender, age, BMI, calcineurin inhibitor (CNI) and MPA (a). Statistical significance was set at P < 0.05. Our endpoints actuarial transplantation-free survival and overall survival showed no missing data. We estimate the missing data for ACR or infectious and biliary complications very low due to the thorough aftercare in the post-transplant follow-up care clinic with standard questionnaires.
Results Baseline characteristics An estimated number of 450 patients has been screened for eligible IS regimen. No patient was excluded for any other reason than a differing immunosuppressant regimen. The final prospective, observational study cohort comprises 117 patients with a 12-month follow-up period. 94 patients were male (80.3%), the median age at inclusion into the study was 56.1 ± 10.8 years, whereas the age at liver transplantation
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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was 50.8 ± 12.0 years. The median time on treatment with immunosuppressants was 6.3 ± 5.2 years and the median time on MMF treatment was 3.2 ± 2.6 years. All patients in our cohort were on concomitant immunosuppression with CNIs. The most common co-immunosuppressant in our cohort was cyclosporine in 53 (45.3%) patients followed by tacrolimus in 40 (34.2%) and prolonged-release tacrolimus in 24 (20.5%) patients (Table 1). Regarding biliary status, most of patients (101 (86.3%)) were transplanted using end-to-end biliary anastomosis, whereas 16 patients (13.7%) received primary biliodigestive diversion. The leading causes of liver transplantation were alcoholic liver cirrhosis (38; 32.5%), chronic viral hepatitis (29; 25.2%) primary sclerosing cholangitis (PSC) (19; 16.5%), followed by cryptogenic cirrhosis (7; 6.0%), autoimmune hepatitis (4; 3.4%), M. Wilson (3; 2.6%), medical-toxic (3; 2.6%).
MPA and IMPDH measurement The MPA levels showed a broad interindividual variability at t(0) (2.0 ± 1.8 ng/ml), t(30) (12.7 ± 9.0 ng/ml) and t(120) (7.5 ± 4.3 ng/ml). Accordingly, IMPDH activity showed large inter individual differences at t(o) (23.2 ± 9.5 nmol/h/mg), at t(30) (16.3 ± 8.8 nmol/h/mg) and t(120) (18.2 ± 8.7 nmol/h/mg) as well. MPA levels and IMPDH activity correlate significantly at t(30) (Pearson’s correlation coefficient: -0.322; P = 0.001) and t(120) (Pearson’s correlation coefficient: -0.157; P = 0.012) (Fig. 1). Body-Mass-Index (BMI) showed a negative correlation with MPA level at all three timepoints (t(0): R = -0.315, P = 0.001; t (30) R = -0,191, P = 0.046; t(120) R = -0,292, P = 0.002). Between BMI and IMPDH activity there was no significant correlation. We found patients with elevated serum creatinine levels having significantly higher MPA baseline levels (Mann-Whitney-U: p =0.044). This is probably due to the postulated effect of concomitant higher blood urea nitrogen (BUN) and Mycophenolic Acid Glucuronide (MPAG), both of which are competing with MPA regarding protein binding to Albumin [16,17]. Considering patients’ co-medication, cyclosporine was associated with lower MPA baseline level. The median MPA baseline level in 53 patients with cyclosporine was 1,28 g/mL in contrast to 2,02 g/mL in 64 patients on tacrolimus (P = 0.02) (Table 2). With regard to IMPDH levels we found no significant influence of concomitant immunosuppression with CNI (Table 3). We further found a marginal effect of ursodeoxycholic acid (UDCA) on MPA levels, with lower levels at baseline and t(30) (Mann-Whitney-U: P = 0.07 and 0.03 respectively). On the contrary, we did not find any influence on MPA levels of proton pump inhibitors (PPI), cholestyramine, ciprofloxacin, amoxicillin/clavulanic acid. Furthermore no differences could be shown due to inflammatory bowel diseases (IBD) or patients staying sober until the t(120) blood sampling.
Infectious and biliary complications Within the prospective observational period 61 (52.1%) patients had reported infectious complications of which 30 (25.6%) patients required hospitalization due to their infection. During the same period 33 (28.2%) patients showed anastomotic bile duct strictures and 9 (7.7%) patients had
non-anastomotic bile duct strictures during the follow-up period. With regard to MPA level we found no correlation with infectious or biliary complications within the follow-up period. Patients with baseline IMPDH(a) below the median had significant more viral infections (17 (29.3%) vs. 6 (10.2%); P = 0.009) with especially more CMV infections (6 (21.4%) vs. 1 (3.4%); P = 0.03)). Furthermore, patients with baseline IMPDH(a) above the median developed more often non-anastomotic biliary strictures (8 (13.6%) vs. 1 (1.7%), P = 0.03). We performed uni- and multivariate analysis including gender, age at transplantation, BMI, CNI co-medication, MPA(AUC) and IMPDH (0) to detect association with infection-, rejection and non-anastomic biliary stricture-free survival. Regarding general infection-free survival we could show a significant association with gender, age at transplantation, BMI, CNI co-medication and MPA(AUC) but not IMPDH(0). Whereas neither IMPDH(0) nor MPA(AUC) showed any association with bacterial infection, IMPDH (0) was significantly associated with viral infections in general (P = 0.04) and CMV-infections especially (P = 0.02). IMPDH(0) and MPA(AUC) showed no association with rejection-free or non-anastomotic biliary stricture-free survival in multivariate analysis.
Clinical course Within the prospective study period 7 patients met an endpoint resulting in 3 (2.6%) deaths and 5 (4.3%) retransplantations (one patient met both endpoints). We found the group reaching the combined clinical endpoint of death and re-transplantation showing significantly lower MPA baseline values and a trend towards lower MPAlevels during the two hours after administration (t(0) 0.9 ± 0.7 vs. 2.1 ± 1.8 g/ml Mann-Whitney-U: P = 0.02; t (30) 6.9 ± 5.0 vs. 13.1 ± 9.2 g/ml P = 0.06; t (120) 5.0 ± 1.4 vs. 7.8 ± 4.5 g/ml; Mann-Whitney-U: P = 0.08). Corresponding IMPDH levels showed no statistically significant difference during the two-hour course after MPA administration. We observed no association between the occurrence of an endpoint and time to transplantation or time on MMF on univariate analysis. Neither did time to transplantation and time on MMF show to be associated with higher or lower MPA or IMPDH values.
Measurement of IMPDH activity with simplified MPA AUC We calculated a simplified MPA(AUC) with the MPA level at baseline, 30 and 120 minutes after MPA administration. The median MPA(AUC) was 42.1 ± 2.0 g/ml*hr. We compared patients with MPA(AUC) above and below the median. Whereas we found no differences with regard to baseline characteristics at entry into the study patients with MPA (AUC) below the median experienced significantly more often the combined clinical endpoint (12.1% (7/58) vs. 0.0% (0/57); P = 0.002) and had a reduced actuarial re-transplantation-free survival (1.0 year vs. 0.58 years; Log-rank: P = 0.007) during the prospective one year followup period. We performed Cox regression analysis including gender, age at OLT, BMI, CNI, MPA(AUC) and IMPDH(a). In univariate Cox regression analysis BMI and MPA(AUC)
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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Figure 1 Table 2
Whole cohort
Cyclosporine (n = 53)
Tacrolimus total (n = 64)
Tacrolimus (n = 40)
Prolonged-release Tacrolimus (n = 24)
2.03 ± 1.76 12.75 ± 9.09 7.58 ± 4.39
1.53 ± 1.03 12.62 ± 9.19 7.91 ± 4.37
2.45 ± 2.12 12.86 ± 9.08 7.32 ± 4.42
2.56 ± 2.25 13.41 ± 9.50 7.51 ± 4.22
2.27 ± 1.94 11.96 ± 8.45 7.00 ± 4.80
IMPDH activity dependent on concomitant immunosuppression.
IMPDH (0) IMPDH (30) IMPDH (120)
Table 4
IMPDH activity vs. corresponding MPA level at t(0), t(30) and t(120).
MPA level dependent on concomitant immunosuppression.
MPA (0) MPA (30) MPA (120)
Table 3
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Whole cohort
Cyclosporine (n = 53)
Tacrolimus total (n = 64)
Tacrolimus (n = 40)
Prolonged-release Tacrolimus (n = 24)
23.26 ± 9.51 16.38 ± 8.84 18.28 ± 8.74
22.34 ± 10.23 14.94 ± 9.64 17.15 ± 8.80
24.01 ± 8.88 17.58 ± 8.00 19.22 ± 8.65
25.76 ± 8.72 18.39 ± 7.77 20.22 ± 9.75
21.09 ± 8.53 16.23 ± 8.36 17.56 ± 6.24
Cox regression analysis.
Risk factor
Sex Age at Transplantation BMI CNI MPA(AUC) IMPDH (a)
Univariate
Multivariate
HR
HR (95% CI)
P-value
HR
HR (95% CI)
P-value
1.1 1.0 0.8 1.2 0.1 0.32
0.7—1.7 0.9—1.0 0,7—1.0 0.4—3.9 0.1—0.6 0.10—1.05
0.6 0.3 0.04 0.8 0.01 0.06
0.1 0.98 0.81 1.03 0.93 0.20
0.0—3.2 0.93—1.03 0.67—0.93 0.26—4.06 0.87—1.00 0.05—0.74
0.4 0.4 0.02 0.9 0.04 0.02
HR, hazard ratio; CI, confidence interval; BMI, Body Mass Index; CNI, Calcineurin Inhibitor; MPA(AUC), mycophenolate acid; IMPDH, Inosine-monophosphat dehydrogenase. Data show prospective factors for longer survival until re-liver transplantation or death. In univariate analysis and multivariate analysis BMI, MPA(AUC) and IMPDH(a) reached significance (P < 0.05).
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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were significantly associated with reduced actuarial retransplantation-free survival. In multivariate analysis BMI, MPA (AUC) and IMPDH(a) were independently associated with reduced actuarial re-transplantation-free survival (Table 4).
Discussion In this observational prospective cohort study, we analyzed MPA level and corresponding IMPDH activity in 117 stable liver transplanted patients. We found patients with higher IMPDH inhibition to be at risk for infectious complications, especially viral infections, including CMV. On the contrary, insufficient IMPDH inhibition after MPA administration was associated with non-anastomotic bile duct strictures and reduced re-transplantation-free survival. The applied abbreviated MPA(AUC) might therefore be a useful tool for individual therapeutic drug monitoring of MPA mediated immunosuppression. Whereas studies have been performed assessing IMPDH in liver transplant patients in general [18] or examining IMPDH in a short-term period up to 3 weeks after liver transplantation [19,20] this—to our knowledge is the first prospective study linking IMPDH to clinical course in stable liver transplant patients with a 1 year follow up. Measurement of MPA levels and IMPDH activity in liver transplanted patients shows a high interpatient variability in our cohort. This is in line with previous reports showing highly variable absorption data in renal allograft recipients [21]. Several factors have shown to contribute to this effect, like interactions with comedications (e.g. proton pump inhibitors or cyclosporine) or changes in renal function or albumin levels [10,22—24]. In our cohort we evaluated several factors and confirmed comedication with cyclosporine and renal function to be associated with MPA levels, probably explained by an at least partly renal excretion of MPA[25]. However, IMPDH levels seems not to be influenced significantly by comedication with CNI in our cohort, probably due to the limited sample size or other confounders. With regard to this, comedication with UDCA also shows marginal influence on MPA levels in our cohort. Another important confounder seems to be BMI. There is a clear inverse correlation of BMI with MPA levels in our cohort. This has especially to be taken into account with regard to gender dependent differences, as women with a lower BMI on average showed higher MPA levels in our cohort. Weight adapted MPA dosing might be an easy and practical idea to adjust individual immunosuppression [26]. Adverse events might be at least in part be explained by inadequate individual MPA dosing [27,28]. There was a clear correlation of higher MPA levels with viral infections, especially CMV infections. Accordingly, we could verify IMPDH level as an independent risk factor for viral infections in our cohort. CMV remains the most common opportunistic infection following solid-organ transplant and might become a cause of life-threatening disease and allograft rejection in liver transplant recipients[29—32]. Adapted dosing of MPA might be a possibility to reduce risk for CMV infection in liver transplanted patients. Despite infections, complications of the biliary system are a major threat of graft and host survival [33—35]. With regard to this we could show that patients with less IMPDH inhibition developed more often non-anastomotic biliary strictures. In contrast to anastomotic strictures, non-anastomotic
strictures seem to be associated with immunological risk factors, that might be prevented by optimized immunosuppression dosing[36—38]. To our knowledge this is the first report that links MPA dependent immunosuppression with this immune mediated complication in liver transplanted patients. While CNI mediated immunosuppression is usually monitored and adjusted according baseline levels this is not true for MPA. Our results supply evidence that despite kidney protection due to CNI reduction immunosuppression with MPA confers several potential effects, that requires individual adaption. Measurement of IMPDH activity might further improve individualization of immunosuppression [39—41]. However, MPA levels, especially when abbreviated AUC was applied correlates well with patient course after liver transplantation and might be easier to establish in a clinical setting than IMPDH activity. Several limitations of the current study have to be taken into account. First, we report about a single center cohort for what reasons center effects might hamper the generality of our findings. In addition, each patient was sampled only once. Hence possible known confounders like for example renal function and albumin concentration could have changed during the prospective follow-up period. However, we were not able to identify any confounder with regard to the baseline clinical or laboratory parameters. The considerable number of patients with different underlying liver disease in our cohort should also prevent selection bias. Second, we only measured MPA levels and corresponding IMPDH activity during once consultation in each patient. As we found high interindividual variations estimation of MPA and IMPDH in repeated intervals might improve reliability of our results. Though, we aimed to apply an easy tool to assess MPA dependent immunosuppression in a feasible manner in an outpatient setting. Furthermore, we only analyzed stable patients with unchanged MPA and CNI dosing for several months. For that reason, we believe our measurements reflects a reliable reference point for MPA levels in our patients. The significant correlation of as well MPA levels and clinical endpoints argue for the validity of out method. This is backed up by the fact that MPA AUC correlates as well and on a higher level of significance with clinical endpoints. Third, MPA AUC was determined by using a limited sampling (LSS) strategy derived in a study of renal transplant patients, who were treated with enteric-coated mycophenolic sodium (EC-MPS) and not MMF [10]. Whereas these patients were on cyclosporine the patients in this cohort received partly tacrolimus and partly cyclosporine. Furthermore, another organ had been transplanted. The active component in both, EC-MPS and MMF, is MPA and the CNI co-medication did not identify as a risk factor in univariate or multivariate analyses. Thereby we believe it is valid to use the LSS derived in the previous mentioned study. In conclusion we found measurement of MPA levels and estimation of corresponding IMPDH activity in liver transplanted patients as a useful instrument for monitoring individual MPA dependent immunosuppression. While patients with higher IMPDH inhibition acquires more often CMV infections, insufficient or delayed IMPDH inhibition after MPA administration is associated with non-anastomotic bile duct strictures and reduced re-transplantation-free survival. These pharmacokinetic and safety data support further research on the hypothesis that adequate MPA
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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Effect of mycophenolic acid in liver transplant patients exposure could improve clinical outcome. Further studies are warranted to analyze adaption of MPA dosing according to individual MPA levels, MPA AUC and IMPDH activity in liver transplanted patients.
Author contributions Manuel C Neuberger: designed research/study, performed research/study, collected data, analyzed data, wrote the paper. Claudia Sommerer: designed research/study, performed research/study, contributed important reagents, provided incubation protocol, provided HPLC protocol and HPLClaboratory, proofread the paper. Sabine Böhnisch: performed HPLC. Nicole Metzendorf: performed HPLC. Arianeb Mehrabi: proofread the paper, collected data. Wolfgang Stremmel: proofread the paper, analyzed data. Daniel Gotthardt: designed research/study, analyzed data, proofread the paper. Martin Zeier: analyzed data, proofread the paper. Karl Heinz Weiss: proofread the paper, analyzed data. Christian Rupp: designed research/study, performed research/study, collected data, analyzed data, wrote the paper.
Funding Supportive foundations: no funding sources.
Ethical statement The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items. Informed consent: all involved gave their informed consent prior to study inclusion.
Disclosure of interest The authors declare that they have no competing interest.
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Clinics and Research in Hepatology and Gastroenterology (2019) xxx, xxx—xxx
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ORIGINAL ARTICLE
Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients M. Neuberger a,1, C. Sommerer b,1, S. Böhnisch b, N. Metzendorf b, A. Mehrabi c, W. Stremmel a, D. Gotthardt a, M. Zeier b, K.H. Weiss a, C. Rupp a,∗ a
University Hospital Heidelberg, Internal Medicine IV, 69120 Heidelberg, Germany University Hospital Heidelberg, Division of Nephrology, 69120 Heidelberg, Germany c University of Heidelberg, Department of General, Visceral, and Transplantation Surgery, 69120 Heidelberg, Germany b
KEYWORDS Liver transplantation; Immunosuppression; IMPDH; Mycophenolic acid; Limited sampling strategy
∗ 1
Summary Background: Due to the development of immunosuppressants, the focus in transplanted patients has shifted from short-term to long-term survival as well as a better adjustment of these drugs in order to prevent over- and under-immunosuppression. Mycophenolic acid (MPA) is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) and approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation, where it has become a part of the standard therapy. Targeting inosine monophosphate IMPDH activity as a surrogate pharmacodynamic marker of MPA-induced immunosuppression may allow a more accurate assessment of efficacy and aid in limiting toxicity in liver transplanted patients. Aim: Assess IMPDH-inhibition in liver transplant recipients and its impact on biliary/infectious complications, acute cellular rejection (ACR) and liver dependent survival. Methods: This observational cohort study comprises 117 liver transplanted patients that were treated with mycophenolate mofetil (MMF) for at least 3 months. Blood samples (BS) were collected and MPA serum level and IMPDH activity were measured before (t(0)), 30 minutes (t(30)) and 2 h after (t(120)) MMF morning dose administration. Regarding MPA, we assessed the area under the curve (AUC). Patients were prospectively followed up for one year and assessed for infectious and biliary complications, episodes of ACR and liver dependent survival. Results: The MPA levels showed a broad interindividual variability at t(0) (2.0 ± 1.8 ng/ml), t(30) (12.7 ± 9.0 ng/ml) and t(120) (7.5 ± 4.3 ng/ml). Corresponding IMPDH activity was at t(o) (23.2 ± 9.5 nmol/h/mg), at t(30) (16.3 ± 8.8 nmol/h/mg) and t(120) (18.2 ± 8.7 nmol/h/mg). With regard to MPA level we found no correlation with infectious or biliary complications
Corresponding author. University Hospital Heidelberg, Department of Gastroenterology, INF 410, 69121 Heidelberg, Germany. E-mail addresses: [email protected], [email protected] (C. Rupp). Both authors contributed equally.
https://doi.org/10.1016/j.clinre.2019.12.001 2210-7401/© 2019 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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M. Neuberger et al. within the follow-up period. Patients with baseline IMPDH(a) below the median had significant more viral infections (6 (10.2%) vs. 17 (29.3%); P = 0.009) with especially more cytomegalovirus (CMV) infections (1 (3.4%) vs. 6 (21.4%); P = 0.03)). Furthermore, patients with baseline IMPDH(a) above the median developed more often non-anastomotic biliary strictures (8 (13.6%) vs. 1 (1.7%), P = 0.03). We found the group reaching the combined clinical endpoint of death and re-transplantation showing significantly lower MPA baseline values (t(0) 0.9 ± 0.7 vs. 2.1 ± 1.8 g/ml Mann-Whitney-U: P = 0.02). We calculated a simplified MPA(AUC) with the MPA level at baseline, 30 and 120 minutes after MPA administration. Whereas we found no differences with regard to baseline characteristics at entry into the study patients with MPA (AUC) below the median experienced significantly more often the combined clinical endpoint (12.1% (7/58) vs. 0.0% (0/57); P = 0.002) and had a reduced actuarial re-transplantation-free survival (1.0 year vs. 0.58 years; Log-rank: P = 0.007) during the prospective one-year followup period. In univariate and multivariate analysis including gender, age, BMI, ACR, MPA (AUC) and IMPDH(a) only BMI, MPA (AUC) and IMPDH(a) were independently associated with reduced actuarial re-transplantation-free survival. Conclusion: MPA-levels and IMPDH-activity in liver transplanted patients allows individual risk assessment. Patients with higher IMPDH inhibition acquire more often viral infections. Insufficient IMPDH inhibition is associated with development of non-anastomotic bile duct strictures and reduced re-transplantation-free survival. © 2019 Elsevier Masson SAS. All rights reserved.
Introduction Due to the development of immunosuppressants the focus in transplanted patients has shifted from short-term to longterm survival as well as a better adjustment of these drugs in order to prevent over- and under-immunosuppression [1,2]. Mycophenolic acid (MPA) is a noncompetitive inhibitor of inosine monophosphate dehydrogenase (IMPDH) and approved for prophylaxis of acute rejection after kidney, heart, and liver transplantation, where it has become a part of the standard therapy [3—6]. Targeting inosine monophosphate IMPDH activity as a surrogate pharmacodynamic marker of MPA-induced immunosuppression may allow a more accurate assessment of efficacy and aid in limiting toxicity in liver transplanted patients [7—9]. We aimed to assess the effect of IMPDH inhibition in liver transplant recipients and evaluate the impact on biliary and infectious complications, episodes of acute cellular rejections (ACR) and liver dependent survival.
Patients and methods
Table 1
Baseline characteristics.
Patients Gender Male Female Age at serum sampling Age at liver transplantation Immunsuppression MMF+ Cyclosporine Tacrolimus Further characteristics BMI [kg/m2 ] Serum bilirubine [mg/dl] Serum creatinine [mg/dl] INR MELD Leucocytes [/nl] Lymphocytes [%] Thrombocytes [/nl] GOT [U/l] GPT [U/l]
117 94 (80.3%) 23 (19.7%) 561 ± 10.8 50.8 ± 12.0 53 (45.3%) 64 (54.7%) 26.8 ± 5.16 1.06 ± 2.08 1.22 ± 0.95 1.05 ± 0.25 9.2 ± 4.03 5.9 ± 2.05 22.6 ± 7.96 186.7 ±74.17 30.4 ± 27.21 32.41 ± 42.01
Study design In this prospective cohort study blood samples (BS) were withdrawn and MPA serum levels and IMPDH activity were measured in 117 stable liver transplant patients. BS were collected from January throughout June 2014 during the patients’ post-transplant follow-up appointments in our outpatient clinic. Therefore, all patients having an appointment in our outpatient department during the study period were screened for MMF + CNI and included consecutively. No patient was excluded for any other reason than a
differing immunosuppressant regimen. ‘‘Stable’’ was defined as being seen in an outpatient setting, not being on steroids or showing clinical or laboratory signs of infection or hepatic decompensation and having a stable CNI dosing resulting in the specified CNI target range. This is mirroring in an average MELD score of 9.2 ± 4.0 and further laboratory markers for infection, kidney and liver function within the normal range (see Table 1). Median time from transplantation was 4.7 ± 5.2 years. Patients admitted to our ward, showing signs of acute rejection
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Effect of mycophenolic acid in liver transplant patients or on prolonged steroid therapy were excluded as well as patients with proven CMV infection. The study patients have already been on MMF for at least 3 months. Blood samples were taken before (t(0)), 30 minutes (t(30)) and 2 hours after (t(120)) MMF morning dose administration. The MMF dosing was 2000 mg/day (87,2%) in vast majority of patients included into the study. This reflects the standard dose rate of MMF in combined IS regimen in stable patients. The residual patients were on 1000 mg/day (10,3%) or had individual MMF dosing (500 mg—3000 mg per day) adjusted to their needs, which were figured out and approached to in post-transplant follow-up, especially balanced between an impaired kidney-function and adverse drug reactions. All patients were on double immunosuppression (IS) consisting of MMF and either cyclosporin A or tacrolimus. Patients were followed up prospectively for one year, starting at the time of sampling, and assessed for infectious and biliary complications, episodes of ACR and liver dependent survival. All ACRs were biopsy-proven and reviewed by an experienced gastrointestinal pathologist, whereas the anastomotic and non-anastomotic biliary strictures were diagnosed endoscopically. The follow-up period was 11 to 16 months. We assessed information in the post-transplant follow-up care clinic, where patients were seen every 3, 6 or 12 months depending on the interval to transplantation and their clinical course. Urgent complications were registered there as well since the post-transplant follow-up care clinic is the first point of contact for this highly selective patient population. Additionally, we assessed the patient’s clinical course by screening the routine follow-up reports as well as the urgent complications in the corresponding electronic patients’ records.
MPA & IMPDH measurement The measurement of MPA levels has been performed by the routine laboratory of the University Hospital Heidelberg using the EMIT (enzyme-multiplied immunoassay technique (EMIT; Dimension XL, Dade Behring, Marburg, Germany)). The isolation and incubation process of peripheric blood monocytic cells (PBMC) and the determination of the IMPDH activity using high performance liquid chromatography (HPLC) have been performed in the laboratories of our working groups, where this method has been used and published before to determine the IMPDH activity in renal transplant patients [10]. As a first step the PBMCs have to be isolated from lithiumheparin whole blood sample. The method we used has initially been established by Böyum et al. in 1968 [11] and was modified and improved by Glander et al. [12,13]: The lithium-heparin whole blood sample was mixed with phosphate buffered saline (PBS) and stacked as a layer on top of Ficoll. After centrifugation that mixture for 20 minutes (room temperature, 2500 rpm) the PBMCs presented in a separate layer and could be taken to another sample tube with the help of a pipette. During this next step the PBMCs were washed with 5 ml of PBS and centrifugated again (room temperature, 2500 rpm). In this way PBMC pellets formed, which were brought into solution with 250 l of HPLC water. These cell lysates were stored at -80 ◦ C.
3 In order to determine the IMPDH-activity next to a chromatographic and a radiometric method there is a method available using real-time polymerase chain reaction (PCR) [14]. The determination of IMPDH activity using HPLC seems the best accessible and most widely used one [15]. Compared to other methods HPLC reduces the variability of results and provides quick results [14,15]. This method’s basis is the reaction of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), which is catalyzed by IMPDH. After incubating the PBMCs with IMP and NAD+ we could quantify XMP using HPLC with subsequent ultraviolet (UV) detection. After determining the protein concentration and knowing the amount of XMP we could calculate the IMPDH activity in ‘‘nmol/h/mg Protein’’. For protein determination we used Thermo Scientific’s ‘‘Pierce BCA Protein Assay Kit (#23225)’’. For IMPDH the values were assessed separately whereas regarding MPA the area under the curve (AUC(0—2 h)) was calculated using a limited sampling strategy (LSS) [10].
Ethical approval Blood withdrawal, data collection and prospective follow-up were performed with approval of the local ethics committee (‘‘Ethikkommission Medizinische Fakultät Heidelberg’’, S-043/2011). Written informed consent was obtained from all volunteers.
Statistical analysis Calculations were carried out using IBM® SPSS® Statistics 20 for Windows (SPSS Inc., Chicago, IL, USA)). Frequencies were compared using the chi-squared test or the Fisher’s exact test, where appropriate. Continuous data were compared using the nonparametric Wilcoxon rank-sum test. Actuarial transplantation-free survival was estimated using a Kaplan-Meier product limit estimator. Differences between the actuarial estimates were tested using the log rank test. Factors that independently affected the risk of reduced transplantation-free survival were determined using Cox proportional hazard ratio models with simultaneous adjustment for gender, age, BMI, calcineurin inhibitor (CNI) and MPA (a). Statistical significance was set at P < 0.05. Our endpoints actuarial transplantation-free survival and overall survival showed no missing data. We estimate the missing data for ACR or infectious and biliary complications very low due to the thorough aftercare in the post-transplant follow-up care clinic with standard questionnaires.
Results Baseline characteristics An estimated number of 450 patients has been screened for eligible IS regimen. No patient was excluded for any other reason than a differing immunosuppressant regimen. The final prospective, observational study cohort comprises 117 patients with a 12-month follow-up period. 94 patients were male (80.3%), the median age at inclusion into the study was 56.1 ± 10.8 years, whereas the age at liver transplantation
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was 50.8 ± 12.0 years. The median time on treatment with immunosuppressants was 6.3 ± 5.2 years and the median time on MMF treatment was 3.2 ± 2.6 years. All patients in our cohort were on concomitant immunosuppression with CNIs. The most common co-immunosuppressant in our cohort was cyclosporine in 53 (45.3%) patients followed by tacrolimus in 40 (34.2%) and prolonged-release tacrolimus in 24 (20.5%) patients (Table 1). Regarding biliary status, most of patients (101 (86.3%)) were transplanted using end-to-end biliary anastomosis, whereas 16 patients (13.7%) received primary biliodigestive diversion. The leading causes of liver transplantation were alcoholic liver cirrhosis (38; 32.5%), chronic viral hepatitis (29; 25.2%) primary sclerosing cholangitis (PSC) (19; 16.5%), followed by cryptogenic cirrhosis (7; 6.0%), autoimmune hepatitis (4; 3.4%), M. Wilson (3; 2.6%), medical-toxic (3; 2.6%).
MPA and IMPDH measurement The MPA levels showed a broad interindividual variability at t(0) (2.0 ± 1.8 ng/ml), t(30) (12.7 ± 9.0 ng/ml) and t(120) (7.5 ± 4.3 ng/ml). Accordingly, IMPDH activity showed large inter individual differences at t(o) (23.2 ± 9.5 nmol/h/mg), at t(30) (16.3 ± 8.8 nmol/h/mg) and t(120) (18.2 ± 8.7 nmol/h/mg) as well. MPA levels and IMPDH activity correlate significantly at t(30) (Pearson’s correlation coefficient: -0.322; P = 0.001) and t(120) (Pearson’s correlation coefficient: -0.157; P = 0.012) (Fig. 1). Body-Mass-Index (BMI) showed a negative correlation with MPA level at all three timepoints (t(0): R = -0.315, P = 0.001; t (30) R = -0,191, P = 0.046; t(120) R = -0,292, P = 0.002). Between BMI and IMPDH activity there was no significant correlation. We found patients with elevated serum creatinine levels having significantly higher MPA baseline levels (Mann-Whitney-U: p =0.044). This is probably due to the postulated effect of concomitant higher blood urea nitrogen (BUN) and Mycophenolic Acid Glucuronide (MPAG), both of which are competing with MPA regarding protein binding to Albumin [16,17]. Considering patients’ co-medication, cyclosporine was associated with lower MPA baseline level. The median MPA baseline level in 53 patients with cyclosporine was 1,28 g/mL in contrast to 2,02 g/mL in 64 patients on tacrolimus (P = 0.02) (Table 2). With regard to IMPDH levels we found no significant influence of concomitant immunosuppression with CNI (Table 3). We further found a marginal effect of ursodeoxycholic acid (UDCA) on MPA levels, with lower levels at baseline and t(30) (Mann-Whitney-U: P = 0.07 and 0.03 respectively). On the contrary, we did not find any influence on MPA levels of proton pump inhibitors (PPI), cholestyramine, ciprofloxacin, amoxicillin/clavulanic acid. Furthermore no differences could be shown due to inflammatory bowel diseases (IBD) or patients staying sober until the t(120) blood sampling.
Infectious and biliary complications Within the prospective observational period 61 (52.1%) patients had reported infectious complications of which 30 (25.6%) patients required hospitalization due to their infection. During the same period 33 (28.2%) patients showed anastomotic bile duct strictures and 9 (7.7%) patients had
non-anastomotic bile duct strictures during the follow-up period. With regard to MPA level we found no correlation with infectious or biliary complications within the follow-up period. Patients with baseline IMPDH(a) below the median had significant more viral infections (17 (29.3%) vs. 6 (10.2%); P = 0.009) with especially more CMV infections (6 (21.4%) vs. 1 (3.4%); P = 0.03)). Furthermore, patients with baseline IMPDH(a) above the median developed more often non-anastomotic biliary strictures (8 (13.6%) vs. 1 (1.7%), P = 0.03). We performed uni- and multivariate analysis including gender, age at transplantation, BMI, CNI co-medication, MPA(AUC) and IMPDH (0) to detect association with infection-, rejection and non-anastomic biliary stricture-free survival. Regarding general infection-free survival we could show a significant association with gender, age at transplantation, BMI, CNI co-medication and MPA(AUC) but not IMPDH(0). Whereas neither IMPDH(0) nor MPA(AUC) showed any association with bacterial infection, IMPDH (0) was significantly associated with viral infections in general (P = 0.04) and CMV-infections especially (P = 0.02). IMPDH(0) and MPA(AUC) showed no association with rejection-free or non-anastomotic biliary stricture-free survival in multivariate analysis.
Clinical course Within the prospective study period 7 patients met an endpoint resulting in 3 (2.6%) deaths and 5 (4.3%) retransplantations (one patient met both endpoints). We found the group reaching the combined clinical endpoint of death and re-transplantation showing significantly lower MPA baseline values and a trend towards lower MPAlevels during the two hours after administration (t(0) 0.9 ± 0.7 vs. 2.1 ± 1.8 g/ml Mann-Whitney-U: P = 0.02; t (30) 6.9 ± 5.0 vs. 13.1 ± 9.2 g/ml P = 0.06; t (120) 5.0 ± 1.4 vs. 7.8 ± 4.5 g/ml; Mann-Whitney-U: P = 0.08). Corresponding IMPDH levels showed no statistically significant difference during the two-hour course after MPA administration. We observed no association between the occurrence of an endpoint and time to transplantation or time on MMF on univariate analysis. Neither did time to transplantation and time on MMF show to be associated with higher or lower MPA or IMPDH values.
Measurement of IMPDH activity with simplified MPA AUC We calculated a simplified MPA(AUC) with the MPA level at baseline, 30 and 120 minutes after MPA administration. The median MPA(AUC) was 42.1 ± 2.0 g/ml*hr. We compared patients with MPA(AUC) above and below the median. Whereas we found no differences with regard to baseline characteristics at entry into the study patients with MPA (AUC) below the median experienced significantly more often the combined clinical endpoint (12.1% (7/58) vs. 0.0% (0/57); P = 0.002) and had a reduced actuarial re-transplantation-free survival (1.0 year vs. 0.58 years; Log-rank: P = 0.007) during the prospective one year followup period. We performed Cox regression analysis including gender, age at OLT, BMI, CNI, MPA(AUC) and IMPDH(a). In univariate Cox regression analysis BMI and MPA(AUC)
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Figure 1 Table 2
Whole cohort
Cyclosporine (n = 53)
Tacrolimus total (n = 64)
Tacrolimus (n = 40)
Prolonged-release Tacrolimus (n = 24)
2.03 ± 1.76 12.75 ± 9.09 7.58 ± 4.39
1.53 ± 1.03 12.62 ± 9.19 7.91 ± 4.37
2.45 ± 2.12 12.86 ± 9.08 7.32 ± 4.42
2.56 ± 2.25 13.41 ± 9.50 7.51 ± 4.22
2.27 ± 1.94 11.96 ± 8.45 7.00 ± 4.80
IMPDH activity dependent on concomitant immunosuppression.
IMPDH (0) IMPDH (30) IMPDH (120)
Table 4
IMPDH activity vs. corresponding MPA level at t(0), t(30) and t(120).
MPA level dependent on concomitant immunosuppression.
MPA (0) MPA (30) MPA (120)
Table 3
5
Whole cohort
Cyclosporine (n = 53)
Tacrolimus total (n = 64)
Tacrolimus (n = 40)
Prolonged-release Tacrolimus (n = 24)
23.26 ± 9.51 16.38 ± 8.84 18.28 ± 8.74
22.34 ± 10.23 14.94 ± 9.64 17.15 ± 8.80
24.01 ± 8.88 17.58 ± 8.00 19.22 ± 8.65
25.76 ± 8.72 18.39 ± 7.77 20.22 ± 9.75
21.09 ± 8.53 16.23 ± 8.36 17.56 ± 6.24
Cox regression analysis.
Risk factor
Sex Age at Transplantation BMI CNI MPA(AUC) IMPDH (a)
Univariate
Multivariate
HR
HR (95% CI)
P-value
HR
HR (95% CI)
P-value
1.1 1.0 0.8 1.2 0.1 0.32
0.7—1.7 0.9—1.0 0,7—1.0 0.4—3.9 0.1—0.6 0.10—1.05
0.6 0.3 0.04 0.8 0.01 0.06
0.1 0.98 0.81 1.03 0.93 0.20
0.0—3.2 0.93—1.03 0.67—0.93 0.26—4.06 0.87—1.00 0.05—0.74
0.4 0.4 0.02 0.9 0.04 0.02
HR, hazard ratio; CI, confidence interval; BMI, Body Mass Index; CNI, Calcineurin Inhibitor; MPA(AUC), mycophenolate acid; IMPDH, Inosine-monophosphat dehydrogenase. Data show prospective factors for longer survival until re-liver transplantation or death. In univariate analysis and multivariate analysis BMI, MPA(AUC) and IMPDH(a) reached significance (P < 0.05).
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were significantly associated with reduced actuarial retransplantation-free survival. In multivariate analysis BMI, MPA (AUC) and IMPDH(a) were independently associated with reduced actuarial re-transplantation-free survival (Table 4).
Discussion In this observational prospective cohort study, we analyzed MPA level and corresponding IMPDH activity in 117 stable liver transplanted patients. We found patients with higher IMPDH inhibition to be at risk for infectious complications, especially viral infections, including CMV. On the contrary, insufficient IMPDH inhibition after MPA administration was associated with non-anastomotic bile duct strictures and reduced re-transplantation-free survival. The applied abbreviated MPA(AUC) might therefore be a useful tool for individual therapeutic drug monitoring of MPA mediated immunosuppression. Whereas studies have been performed assessing IMPDH in liver transplant patients in general [18] or examining IMPDH in a short-term period up to 3 weeks after liver transplantation [19,20] this—to our knowledge is the first prospective study linking IMPDH to clinical course in stable liver transplant patients with a 1 year follow up. Measurement of MPA levels and IMPDH activity in liver transplanted patients shows a high interpatient variability in our cohort. This is in line with previous reports showing highly variable absorption data in renal allograft recipients [21]. Several factors have shown to contribute to this effect, like interactions with comedications (e.g. proton pump inhibitors or cyclosporine) or changes in renal function or albumin levels [10,22—24]. In our cohort we evaluated several factors and confirmed comedication with cyclosporine and renal function to be associated with MPA levels, probably explained by an at least partly renal excretion of MPA[25]. However, IMPDH levels seems not to be influenced significantly by comedication with CNI in our cohort, probably due to the limited sample size or other confounders. With regard to this, comedication with UDCA also shows marginal influence on MPA levels in our cohort. Another important confounder seems to be BMI. There is a clear inverse correlation of BMI with MPA levels in our cohort. This has especially to be taken into account with regard to gender dependent differences, as women with a lower BMI on average showed higher MPA levels in our cohort. Weight adapted MPA dosing might be an easy and practical idea to adjust individual immunosuppression [26]. Adverse events might be at least in part be explained by inadequate individual MPA dosing [27,28]. There was a clear correlation of higher MPA levels with viral infections, especially CMV infections. Accordingly, we could verify IMPDH level as an independent risk factor for viral infections in our cohort. CMV remains the most common opportunistic infection following solid-organ transplant and might become a cause of life-threatening disease and allograft rejection in liver transplant recipients[29—32]. Adapted dosing of MPA might be a possibility to reduce risk for CMV infection in liver transplanted patients. Despite infections, complications of the biliary system are a major threat of graft and host survival [33—35]. With regard to this we could show that patients with less IMPDH inhibition developed more often non-anastomotic biliary strictures. In contrast to anastomotic strictures, non-anastomotic
strictures seem to be associated with immunological risk factors, that might be prevented by optimized immunosuppression dosing[36—38]. To our knowledge this is the first report that links MPA dependent immunosuppression with this immune mediated complication in liver transplanted patients. While CNI mediated immunosuppression is usually monitored and adjusted according baseline levels this is not true for MPA. Our results supply evidence that despite kidney protection due to CNI reduction immunosuppression with MPA confers several potential effects, that requires individual adaption. Measurement of IMPDH activity might further improve individualization of immunosuppression [39—41]. However, MPA levels, especially when abbreviated AUC was applied correlates well with patient course after liver transplantation and might be easier to establish in a clinical setting than IMPDH activity. Several limitations of the current study have to be taken into account. First, we report about a single center cohort for what reasons center effects might hamper the generality of our findings. In addition, each patient was sampled only once. Hence possible known confounders like for example renal function and albumin concentration could have changed during the prospective follow-up period. However, we were not able to identify any confounder with regard to the baseline clinical or laboratory parameters. The considerable number of patients with different underlying liver disease in our cohort should also prevent selection bias. Second, we only measured MPA levels and corresponding IMPDH activity during once consultation in each patient. As we found high interindividual variations estimation of MPA and IMPDH in repeated intervals might improve reliability of our results. Though, we aimed to apply an easy tool to assess MPA dependent immunosuppression in a feasible manner in an outpatient setting. Furthermore, we only analyzed stable patients with unchanged MPA and CNI dosing for several months. For that reason, we believe our measurements reflects a reliable reference point for MPA levels in our patients. The significant correlation of as well MPA levels and clinical endpoints argue for the validity of out method. This is backed up by the fact that MPA AUC correlates as well and on a higher level of significance with clinical endpoints. Third, MPA AUC was determined by using a limited sampling (LSS) strategy derived in a study of renal transplant patients, who were treated with enteric-coated mycophenolic sodium (EC-MPS) and not MMF [10]. Whereas these patients were on cyclosporine the patients in this cohort received partly tacrolimus and partly cyclosporine. Furthermore, another organ had been transplanted. The active component in both, EC-MPS and MMF, is MPA and the CNI co-medication did not identify as a risk factor in univariate or multivariate analyses. Thereby we believe it is valid to use the LSS derived in the previous mentioned study. In conclusion we found measurement of MPA levels and estimation of corresponding IMPDH activity in liver transplanted patients as a useful instrument for monitoring individual MPA dependent immunosuppression. While patients with higher IMPDH inhibition acquires more often CMV infections, insufficient or delayed IMPDH inhibition after MPA administration is associated with non-anastomotic bile duct strictures and reduced re-transplantation-free survival. These pharmacokinetic and safety data support further research on the hypothesis that adequate MPA
Please cite this article in press as: Neuberger M, et al. Effect of mycophenolic acid on inosine monophosphate dehydrogenase (IMPDH) activity in liver transplant patients. Clin Res Hepatol Gastroenterol (2019), https://doi.org/10.1016/j.clinre.2019.12.001
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Effect of mycophenolic acid in liver transplant patients exposure could improve clinical outcome. Further studies are warranted to analyze adaption of MPA dosing according to individual MPA levels, MPA AUC and IMPDH activity in liver transplanted patients.
Author contributions Manuel C Neuberger: designed research/study, performed research/study, collected data, analyzed data, wrote the paper. Claudia Sommerer: designed research/study, performed research/study, contributed important reagents, provided incubation protocol, provided HPLC protocol and HPLClaboratory, proofread the paper. Sabine Böhnisch: performed HPLC. Nicole Metzendorf: performed HPLC. Arianeb Mehrabi: proofread the paper, collected data. Wolfgang Stremmel: proofread the paper, analyzed data. Daniel Gotthardt: designed research/study, analyzed data, proofread the paper. Martin Zeier: analyzed data, proofread the paper. Karl Heinz Weiss: proofread the paper, analyzed data. Christian Rupp: designed research/study, performed research/study, collected data, analyzed data, wrote the paper.
Funding Supportive foundations: no funding sources.
Ethical statement The authors have read the STROBE Statement—checklist of items, and the manuscript was prepared and revised according to the STROBE Statement—checklist of items. Informed consent: all involved gave their informed consent prior to study inclusion.
Disclosure of interest The authors declare that they have no competing interest.
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