Tacrolimus Therapeutic Drug Monitoring in Vietnamese Renal Transplant Recipients

Tacrolimus (Tac) is a potent immunosuppressant drug that is well established for primary immunosuppression in kidney transplantation. It was introduced in the late 1980s as an alternative to cyclosporine-A for the prevention of graft rejection following solid organ transplantation.1 Due to a narrow therapeutic index and its large interpatient and intra-patient pharmacokinetic variability, therapeutic drug monitoring (TDM) is routinely performed for individualization of the Tac dose to maintain drug efficacy and minimize the consequences of overexposure.2 Nowadays, the most frequently used method of Tac monitoring is the measurement of the trough concentration (C0) in whole blood.


INTRODUCTION
Tacrolimus (Tac) is a potent immunosuppressant drug that is well established for primary immunosuppression in kidney transplantation. It was introduced in the late 1980s as an alternative to cyclosporine-A for the prevention of graft rejection following solid organ transplantation. 1 Due to a narrow therapeutic index and its large interpatient and intra-patient pharmacokinetic variability, therapeutic drug monitoring (TDM) is routinely performed for individualization of the Tac dose to maintain drug efficacy and minimize the consequences of overexposure. 2 Nowadays, the most frequently used method of Tac monitoring is the measurement of the trough concentration (C0) in whole blood.
Therapeutic ranges of Tacrolimus have been changed remarkably. In 1995, Lake Louise Consensus Conference recommend preliminary target range of 5 to 20 ng/mL. 3 Tacrolimus trough concentrations were targeted at 3-7 ng/mL with Symphony-Elite. 4 Recently, the European experts on tacrolimus (2009) have proposed new target ranges of trough concentration based on both drug combination and post-transplantation delay. In regiment with mycophenolate mofetil (MMF) and steroid minimization, tacrolimus whole blood concentrations should range from10 to 15 ng/ml during the first 3 months after transplantation, 8 -12 ng/ml in the next period (3 months-12 months), and from 5 ng/ml to 10 ng/ml thereafter. 5 There were studies have evaluated the trough level tacrolimus according to time post-transplantation as well as efficacy and safety within those targets. 4,6 To now, there have been no reports of such studies performed on Vietnamese kidney transplant recipients. Our study aimed to simple description real Tacrolimus therapeutic drug monitoring and determine the outcomes in Vietnamese adult de novo renal transplant recipients.

MATERIALS AND METHODS
A retrospective study was carried out at Department of Renal and Haemodialysis, Military Hospital 103, including one hundred and fourteen Vietnamese patients undergoing renal transplantation. Data of patients were obtained between August 2012 and March 2018. Patients between the ages of 18 and 75 years who received the first single-organ renal transplant from either a living donor or a deceased donor were eligible. Exclusion criteria were paediatric kidney transplant recipients, retransplant or multi-organ transplant patients.

Immunosuppressant protocol used
The immunosuppressive protocol followed in our institution consisted of a triple drug regimen consisting of tacrolimus, mycophenolate mofetil (MMF) and steroids. Induction therapy included basiliximab (Simulect®, Novartis) pre-and 4 days postoperatively and 500 mg intravenous (IV) methyl prednisolone (Solu Medrol®: Pfizer) pre and 12 h postoperatively. Oral tacrolimus (Prograf®, Astellas Pharma) was started preoperatively with a dose of 0.1 mg/kg/day administered in two divided doses. The doses were adjusted in the range therapy followed European consensus conference (2009) or occurrence of the event of adverse effects, rejection. Mycophenolate mofetil (Cellcept®, Roche) was started with tacrolimus at a dose of 1g twice a day and adjusted to lower doses in presence of diarrhea or infection. The next IV dose of steroid decreased by a half in consecutive days to 40 mg/day within one to two weeks' post-transplant. Oral prednisolone (15 mg/day) was initiated right after and was tapered every week to maintaining period of 5 mg/day.

Tacrolimus measurements
Tac whole blood concentrations were determined using chemiluminscent microparticle immunoassay (CMIA, analysed on the Architect system, Abbott Diagnostics, IL, USA). The assay's detection limit was 0.8 ng/ml. Tac trough concentrations were collected prior to the morning dose. Frequency tacrolimus assays were three times weekly the first two weeks, once every week in the third week and fourth week and then at each patient visit during following months. Samples for determination of tacrolimus blood levels were subdivided according to the posttransplantation period into three groups: 0-3 months (G1), 3-12 months (G2) and over 1 year (G3).

Clinical outcomes
Efficacy end points included renal function as indicated by the calculated eGFR during the study, acute rejection (AR), overall patient survival. The eGFR were calculated from serum creatinine measures with the use of the Modification of Diet in Renal Disease formula. 7 Safety was evaluated by clinical assessment including vital signs and laboratory analyses designed to determine the incidence of adverse events. The common adverse events were neurotoxicity, new-onset diabetes (NODAT), post-transplant viral infections, and gastrointestinal disorders, chronic kidney disease and Tac-inducted nephrotoxicity.
NODAT was defined by the American Diabetes Association (ADA) diagnostic criteria 8 . Patients were diagnosed NODAT based plasma glucose criteria, either fasting plasma glucose ≥ 126 mg/dL (7.0 mmol/L) or 2-h plasma glucose ≥ 200 mg/dL (11.1 mmol/L) during oral glucose tolerance test or A1C ≥ 6.5% or in a patient with classic symptoms of hyperglycemia crisis, a random plasma glucose ≥ 200 mg/ dL (11.1 mmol/L).
The blood sample were obtained from recipients who were suspected to have CMV, BK infection upon clinical presentation, physical examination and laboratory results. The disease was diagnosed according to the clinical features and quantitative polymerase chain reaction (qPCR) (and/or CMV antigenemia tests). Viral loads over 10 000 copies/mL for BKV and over 1000 copies/mL for CMV were considered clinically relevant.
The diagnosis of chronic HBV infection was based upon the persistence of hepatitis B surface antigen (HBsAg) for more than 6 months after transplantation. The diagnosis of chronic HBV infection was made in a patient with a positive anti hepatitis C virus test (anti HCV) and positive molecular test that detects the presence of HCV RNA after transplantation.
Chronic kidney disease (CKD) was defined as being present if the glomerular filtration rate (GFR) was < 60 mL/min/1.73 m 2 or evidence of kidney damage such as albuminuria or abnormal findings on renal imaging have been present for 3 months or more.

Statistical analyses
Data management and statistical analyses were carried out using RStudio sofware. Variable distribution was determined with the Shapiro-Wilk test. The Anova test was used to compare normally distributed variables, and the Mann-Whitney U test was used to compare non-normally distributed variables. Variables with a normal distribution were expressed as the mean ± standard deviation (SD) and those with an abnormal distribution were expressed as the median and their interquartile range (IQR). Differences in proportions were analyzed using Fisher's exact test. Time to all events, acute rejection (AR), cytomegalovirus (CMV) and chronic kidney disease (CKD) were analysed by the Kaplan-Meier method.  Table 1.

Dosage of immunosuppressive drugs
Tacrolimus doses in three groups are presented in Table 2. In our population, median tacrolimus doses were reduced after transplantation (0.1 tapered to 0.07 mg/kg/day). Daily dose of mycophenolate mofetil were 1.87 ± 0.3 g.

Tacrolimus concentrations
A total of 3037 blood samples for the determination of Tac concentrations were recorded. The mean number of samples per patient was 26.6 (range 4 to 60). Using a therapeutic range of European consensus conference (2009), 33.3% tacrolimus concentrations were within target ranges in total and this proportion increased over time with the highest proportion in G3 (57%) compared with those in G1     Acute rejection: Three biopsy-proven acute rejections (2.6%) were reported during the study and all of them were in the first three months after transplantation (2 patients were cellular rejection and one case was humoral rejection). Base on Improving Global Outcomes (KDIGO) clinical practice guidelines, three AR patients were high-risk for rejection because of having one or more risk factors for acute rejection include more human leukocyte antigen (HLA) mismatches (2 patients with 6/6 mismatches), positive panel reactive antibody (PRA) Class II and delayed onset of graft function (1 patient).

Patient survival:
Overall survival rates for patients were 98.2%. Of the 2 deaths reported, 1 was caused by AR coexisting CMV pneumonia and the other was due to multi-organ CMV infection coexisting cerebral stroke and HCV.
Safety: Serious adverse events were neurotoxicity, new-onset diabetes (NODAT), post-transplant viral infections, chronic kidney disease (CKD), Tac-associated nephrotoxicity and gastrointestinal disorders Table 3). While neurotoxicity and NODAT only occurred early in the study (G1), the others were found in later periods. Cumulative hazard of all events and some separated outcomes were estimated by Kaplan-Meier analysis (Figure 2).
The incidence of post-transplant diabetes mellitus was 4.4 % (5 patients) and all of them had to convert to cyclosporine.
Overall, incidence of post-transplant viral infections including CMV, BKV, HBV and HCV were 6.1 %, 3.5 %, 6.1 % and 2.6%, respectively. In CMV patients, an organ disease was observed primarily (lungs =5; kidney =1) and 1 case of multi-organ infection. All four patients with BKV developed chronic kidney disease and two of these cases had to switch to cyclosporine. In addition to 4 patients with BKV, chronic kidney disease has been detected in two other cases.
Tac-associated nephrotoxicity was observed with proven biopsy in two patients. One patient with prolonged gastrointestinal disorders changed medication from Tac to cyclosporine.

DISCUSSION
Monitoring blood tacrolimus concentrations is considered necessary in maintaining drug efficacy and minimize the consequences of overexposure. 5 Most previous studies focused on therapeutic drug monitoring of tacrolimus in renal transplant recipients and assessed the clinical response of patients. 9,10 However, only few studies have evaluated the trough tacrolimus according to time post-transplantation as well as efficacy and safety within those targets. 4,6 The primary objective of our study was to summary simply the trough of tacrolimus according to post-transplantation period, as defined by the last European consensus conference (2009) and determine the outcomes in Vietnamese adult renal transplant recipients.
Our results showed that only 33.3% tacrolimus concentrations were within target ranges and 48% of trough blood levels were underthreshold. These results are agreeable with previous observation in Tunisian kidney transplant recipients had similar result. 11 More than 50% of patients were outside the target range. The major reason is presumably the great inter-individual and intra-individual variation of drug levels. 12 However, in some cases it could be attributed directed to an adverse event or to lack of efficacy once a patient has experienced acute rejection. These findings re-emphasized necessity of therapeutic drug monitoring (TDM) in renal transplant recipients.
In our population, the median daily dose was 0.1 (0.09 -0.12) mg/kg in first 3 months after transplantation and this dose reduced by time after transplantation. Some previous had also similar results. In a study performed on a American population, Narayanan et al. 10 showed that the dose in the first month, month 6 and 12 after transplantation were 0. 13  been consistently demonstrated that CYP3A5 expressers (CYP3A5*1 carriers) had a lower dose-adjusted C0 and higher clearance, thus requiring higher tacrolimus doses in order to reach the same steadystate C0 when compared with CYP3A5 non-expressers (CYP3A5*3/*3 carriers). Some studies found that expressers require approximately double the starting dose of tacrolimus. 14  The incidence of acute rejection was 2.6 %. Our proportion are low and similar or lower than those in other reports with a similar followup. 18, 19 Over the last two decades, there have been dramatic reductions in the incidence of AR related to the introduction of more effective immunosuppressive medications (such as mycophenolate mofetil), robust induction regimes and desensitization.
Development of NODAT has been associated with Tac exposure. 20 NODAT increases the risk of cardiovascular events, graft failure and mortality. 21 In our study, 4.4% developed NODAT within three months after transplantation. This low frequency is comparable to those in the Symphony study (10.6% in the first year) and Kamar'study. 4,22 These percentages may roughly be compared since most cases of NODAT often occurs in the first 3 post-transplantation months. 23  In our study, the incidence of opportunistic infections including CMV, BKV, HBV and HCV were low and similar or lower than those in other reports. [24][25][26][27] We hypothesized that low proportion of post-transplant infections may be partly due to our lower Tac levels and careful pretransplant screening, immunization, and post-transplant prophylactic