Highly sensitive and rapid determination of tacrolimus in peripheral blood mononuclear cells by liquid chromatography-tandem mass spectrometry.

After solid organ transplantation, tacrolimus is given to prevent rejection. Therapeutic drug monitoring is used to reach target concentrations of tacrolimus in whole blood. Because the site of action of tacrolimus is the lymphocyte, and tacrolimus binds ~80% to erythrocytes, the intracellular tacrolimus concentration in lymphocytes is possibly more relevant. For this purpose, we aimed to develop, improve and validate a UPLC-MS/MS method to measure tacrolimus concentrations in isolated peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using a Ficoll separation technique, followed by a washing step using red blood cell lysis. A cell suspension of 50 µL containing 1 million PBMCs was used in combination with MagSiMUS-TDMPREP . To each sample we added 30 µL lysis buffer, 20 µL reconstitution buffer containing 13 C2 H4 -tacrolimus as internal standard, 40 µL MagSiMUS-TDMPREP Type I Particle Mix and 175 µL Organic Precipitation Reagent VI for methanol-based protein precipitation. A 10 µL aliquot of the supernatant was injected into the UPLC-MS/MS system. The method was validated, resulting in high sensitivity and specificity. The method was linear (r2  = 0.997) over the range 5.0-1250 pg/1 × 106 PBMCs. The inaccuracy was <5% and the imprecision was <15%. The washing steps following Ficoll isolation could be performed at either room temperature or on ice, with no effect of the temperature on the results. A method for the analysis of tacrolimus concentrations in PBMCs was developed and successfully validated. Further research will be performed to investigate the correlation between concentrations in PBMCs and clinical outcome.

Conversion to Once-Daily Tacrolimus Results in Increased p38MAPK Phosphorylation in T Lymphocytes of Kidney Transplant Recipients.

BACKGROUND: The once-daily formulation of tacrolimus (TAC(OD)) has been developed to overcome adherence problems. Conversion from the twice-daily TAC (TAC(BID)) formulation to TAC(OD) on a 1:1 basis, however, often leads to a decrease of TAC predose concentrations, which averages ∼15%. Switching between the two TAC formulations may thus influence drug efficacy and necessitates therapeutic drug monitoring. As an additional tool in transplantation diagnostics, phospho-specific flow cytometry was used to study the biological effects of conversion on p38MAPK phosphorylation, a kinase involved in T-lymphocyte activation. METHODS: Stable renal transplant recipients (n = 12), at least 1 year after their transplantation, were converted from TAC(BID) to TAC(OD) on 1:1 mg for mg base. Comedication consisted of mycophenolate mofetil (n = 10) and prednisolone (n = 3). TAC whole-blood predose concentrations were determined by immunoassay before and 3 months after conversion. P38MAPK phosphorylation was measured in T lymphocytes by whole-blood phospho-specific flow cytometry. RESULTS: Three months after conversion, no significant decreases in TAC predose concentrations (C0) were found (P = 0.54), whereas p38MAPK phosphorylation increased with 11.4% (P < 0.05) in CD4 and with 15.6% (P < 0.05) in CD8 T lymphocytes. The TAC C0 during treatment with TAC(BID) correlated inversely with p38MAPK phosphorylation in T lymphocytes (rs = -0.638; P < 0.05). CONCLUSIONS: These results suggest that the measurement of p38MAPK phosphorylation status in T lymphocytes is a sensitive method to determine the biological effects of TAC before and after conversion from TAC(BID) to TAC(OD). This method could be a more sensitive tool for therapeutic drug monitoring of TAC.

Pretransplant Tacrolimus Dose Requirements Predict Early Posttransplant Dose Requirements in Blood Group AB0-Incompatible Kidney Transplant Recipients.

BACKGROUND: The aim of this study was to investigate whether pretransplant tacrolimus (Tac) dose requirements of patients scheduled to undergo living donor kidney transplantation correlate with posttransplantation dose requirements. METHODS: The predictive value of Tac dose requirements (defined as the ratio of the Tac predose concentration, C0, divided by the total daily Tac dose, D) pretransplantation on this same parameter posttransplantation was assessed retrospectively in a cohort of 57 AB0-incompatible kidney transplant recipients. These patients started immunosuppressive therapy 14 days before transplant surgery. All patients were using a stable dose of glucocorticoids and were at steady-state Tac exposure before transplantation. RESULTS: Tac dose requirements immediately before transplantation (C0/Dbefore) explained 63% of the Tac dose requirements on day 3 after transplantation: r = 0.633 [F (1, 44) = 75.97, P < 0.01]. No other clinical and demographic variables predicted Tac dose requirements early after transplantation. CONCLUSIONS: Steady-state Tac dose requirement before transplantation largely predicted posttransplantation Tac dose requirements in AB0-incompatible kidney transplant recipients. The importance of this finding is that the posttransplantation Tac dose can be individualized based on a patient's pretransplantation Tac concentration/dose ratio. Pretransplant Tac phenotyping therefore has the potential to improve transplantation outcomes.

A high intrapatient variability in tacrolimus exposure is associated with poor long-term outcome of kidney transplantation.

Tacrolimus is a critical dose drug with a considerable intrapatient variability (IPV) in its pharmacokinetics. We investigated whether a high IPV in tacrolimus exposure is associated with adverse long-term renal transplantation outcomes. Tacrolimus IPV was calculated from predose concentrations measured between 6 and 12 months post-transplantation of 808 renal transplant recipients (RTRs) transplanted between 2000 and 2010. One hundred and eighty-eight (23.3%) patients reached the composite end point consisting of graft loss, late biopsy-proven rejection, transplant glomerulopathy, or doubling of serum creatinine concentration between month 12 and the last follow-up. The cumulative incidence of the composite end point was significantly higher in patients with high IPV than in patients with low IPV (hazard ratio: 1.41, 95% CI: 1.06-1.89; P = 0.019). After the adjustment for several factors, the higher incidence of the composite end point for RTRs with a high IPV remained statistically significant (hazard ratio: 1.42, 95% CI: 1.06-1.90; P = 0.019). Younger recipient age at transplantation, previous transplantation, worse graft function (at month 6 post-transplantation), and low mean tacrolimus concentration at 1 year post-transplantation were additional predictors for worse long-term transplant outcome. A high tacrolimus IPV is an independent risk factor for adverse kidney transplant outcomes that can be used as an easy monitoring tool to help identify high-risk RTRs.

Conversion from twice-daily to once-daily tacrolimus does not reduce intrapatient variability in tacrolimus exposure.

BACKGROUND: Intrapatient variability (IPV) in tacrolimus exposure is associated with renal allograft failure. The aim of this study was to investigate whether conversion from the twice-daily tacrolimus formulation (Tac-TD) to a once-daily formulation (Tac-OD) leads to a lower IPV in tacrolimus exposure. METHODS: Two hundred forty-seven stable renal transplant recipients were converted from Tac-TD to Tac-OD (Advagraf) on a 1:1-mg total daily dose basis. After conversion, patients were followed for 12 months and tacrolimus predose whole-blood concentrations (C0), serum creatinine, estimated glomerular filtration rate, and proteinuria were measured. These parameters were compared with those collected at all outpatient visits in the 12-month period (±3 months) before conversion (Tac-TD period). The IPV was calculated based on the dose-adjusted tacrolimus C0. RESULTS: The Tac-OD formulation provided an excellent graft survival (100%), a low acute rejection rate (0.8%), and good tolerability. Renal function remained stable: estimated glomerular filtration rate 48 (16-90) versus 46 (12-90) mL/min (P = 0.15) before and after conversion, respectively. After conversion to Tac-OD, mean C0 was significantly lower, decreasing from 5.7 ± 1.5 to 5.0 ± 1.5 ng/mL, corresponding to a 12% reduction (P < 0.01). Both drugs had similar IPVs (Tac-TD: 17.3% ± 1.6% versus Tac-OD: 16.4% ± 1.6%, P = 0.31). CONCLUSIONS: Although conversion from Tac-TD to Tac-OD significantly reduces tacrolimus exposure as measured by C0 and seems safe, it does not reduce IPV in tacrolimus exposure.

Clinical implementation of pharmacogenetics in kidney transplantation: calcineurin inhibitors in the starting blocks.

Pharmacogenetics has generated many expectations for its potential to individualize therapy proactively and improve medical care. However, despite the huge amount of reported genetic associations with either pharmacokinetics or pharmacodynamics of drugs, the translation into patient care is still slow. In fact, strong evidence for a substantial clinical benefit of pharmacogenetic testing is still limited, with a few exceptions. In kidney transplantation, established pharmacogenetic discoveries are being investigated for application in the clinic to improve efficacy and to limit toxicity associated with the use of immunosuppressive drugs, especially the frequently used calcineurin inhibitors (CNIs) tacrolimus and ciclosporin. The purpose of the present review is to picture the current status of CNI pharmacogenetics and to discuss the most promising leads that have been followed so far.

Do Asian renal transplant patients need another mycophenolate mofetil dose compared with Caucasian or African American patients?

Mycophenolate mofetil (MMF) is used to prevent acute rejection following solid organ transplantation in transplant centers all over the world. Patients from different ethnic backgrounds are treated with this drug, for which therapeutic drug monitoring (TDM) has not become the standard of practice in most centers. Whether or not some ethnic groups require a different MMF dose has been a topic of debate in recent years. In this review, it is shown that Asian patients, compared with Caucasian patients, with a comparable MMF dose reach higher mycophenolic acid (MPA) exposure. Also clinical experience points toward more adverse events in case of treatment with 1 g MMF bid in Asian patients, and therefore, for this ethnic group, a lower maintenance dose seems justified. In contrast, African American patients reach similar drug concentrations as Caucasians patients receiving the same MMF dose, but due to immunological reasons, they require a higher MMF dose to reach comparable acute rejection incidences. When TDM is performed, clinicians can correct the dose and compensate for interethnic differences in drug exposure. Otherwise, it is important to choose the right dose. This optimal dose is 20-46% lower in Asian transplant recipients than in Caucasian or African American patients.

CYP3A5 and ABCB1 polymorphisms in living donors do not impact clinical outcome after kidney transplantation.

AIM: To investigate the association between donor CYP3A5 and ABCB1 polymorphisms and tacrolimus (Tac)-induced nephrotoxicity and renal function in kidney transplant recipients. METHODS: The CYP3A5 6986A>G and ABCB1 3435C>T polymorphisms were determined in 237 recipients and donors. RESULTS: There was no significant association between Tac-related nephrotoxicity and donor CYP3A5 and ABCB1 genotype. The donor ABCB1 3435C>T polymorphism was associated with estimated glomerular filtration rate on day 7 and month 1. The combined donor-recipient ABCB1 genotype (3435C>T polymorphism) was significantly related with estimated glomerular filtration rate on day 3 and 7 in univariate analysis. However, these differences were no longer statistically significant in multivariate analysis. CONCLUSION: A genetic analysis of ABCB1 and CYP3A5 of kidney transplant donors is not helpful to improve renal transplant outcomes.

Intrapatient Variability in Tacrolimus Exposure Does Not Predict The Development of Cardiac Allograft Vasculopathy After Heart Transplant.

OBJECTIVE: A high intrapatient variability in tacrolimus exposure is associated with poor long-term outcomes after kidney transplant. We hypothesized that a high intrapatient variability of tacrolimus exposure after heart transplant may be associated with cardiac allograft vasculopathy as a determinant of long-term survival of heart transplant recipients. MATERIALS AND METHODS: Eighty-six heart transplant recipients were included. Patients underwent coronary angiography at years 1 and 4 after transplant and were divided according to low and high intrapatient variability of tacrolimus exposure, with the median variability as cut-off. The primary outcome was the association between tacrolimus intrapatient variability and the progression of cardiac allograft vasculopathy score between years 1 and 4. Secondary outcome was this association with acute cellular rejection. RESULTS: There was no significant difference in the proportion of patients with high tacrolimus intrapatient variability in the group that progressed to higher grades of cardiac allograft vasculopathy (n = 15) versus the group without progression (n = 71) at 4-year follow-up (60.0% vs 47.9%; P = .57). There was no significant difference in the proportion of patients with high tacrolimus intrapatient variability between the 58 patients with 1 or more acute cellular rejection episodes and the 28 patients without rejection (51.7% vs 46.4%; P = .82). CONCLUSIONS: A high intrapatient variability in tacrolimus exposure does not appear to influence heart transplant outcomes, unlike its influence on kidney transplant function. A higher immunosuppression exposure after heart transplant, including the use of prednisone often in a combination of 3 immunosuppressive drugs, may protect against the effects of high intrapatient tacrolimus variability.

Overweight Kidney Transplant Recipients Are at Risk of Being Overdosed Following Standard Bodyweight-Based Tacrolimus Starting Dose.

BACKGROUND: Bodyweight-based dosing of tacrolimus (Tac) is considered standard care, even though the available evidence is thin. An increasing proportion of transplant recipients is overweight, prompting the question if the starting dose should always be based on bodyweight. METHODS: For this analysis, data were used from a randomized-controlled trial in which patients received either a standard Tac starting dose or a dose that was based on CYP3A5 genotype. The hypothesis was that overweight patients would have Tac overexposure following standard bodyweight-based dosing. RESULTS: Data were available for 203 kidney transplant recipients, with a median body mass index (BMI) of 25.6 (range, 17.2-42.2). More than 50% of the overweight or obese patients had a Tac predose concentration above the target range. The CYP3A5 nonexpressers tended to be above target when they weighed more than 67.5 kg or had a BMI of 24.5 or higher. Dosing guidelines were proposed with a decrease up to 40% in Tac starting doses for different BMI groups. The dosing guideline for patients with an unknown genotype was validated using the fixed-dose versus concentration controlled data set. CONCLUSIONS: This study demonstrates that dosing Tac solely on bodyweight results in overexposure in more than half of overweight or obese patients.

Intra-patient variability in tacrolimus exposure: causes, consequences for clinical management.

Tacrolimus (Tac) is widely used for the prevention of rejection after solid organ transplantation. Finding the optimal balance between effective Tac concentrations and toxicity is a challenge and requires therapeutic drug monitoring. In addition to the well-known inter-patient variability, the clinical use of Tac is also complicated by considerable intra-patient variability (IPV) in Tac exposure. Tac IPV is defined as the amount of fluctuation of whole-blood concentrations over a certain period of time during which the Tac dose remains unchanged. A high IPV in Tac exposure has recently been recognized as a strong risk factor for acute rejection and poor long-term kidney transplantation outcome. In addition to non-adherence, several other factors determine the magnitude of the IPV in Tac exposure. Quantification of IPV is easy and can be easily incorporated into everyday clinical practice as a tool for optimizing transplantation outcomes.

ATP-binding cassette transporters as pharmacogenetic biomarkers for kidney transplantation.

Immunosuppressive drugs used in organ transplantation are highly effective in preventing acute rejection. However, the clinical use of these drugs is complicated by the fact that they display highly variable pharmacokinetics and pharmacodynamics between individual patients. The influence of genetic variation on the interindividual variability in immunosuppressive drug disposition, efficacy, and toxicity has been explored in recent years. The polymorphically-expressed ATP-binding cassette (ABC) transporter proteins, in particular ABCB1 and ABCC2, have been investigated extensively because they play an important role in the absorption, distribution and elimination of many immunosuppressive drugs in use today. From these studies it can be concluded that polymorphisms in ABCB1 and ABCC2 have no consistent effect on immunosuppressant pharmacokinetics and toxicity although polymorphisms in ABCB1 appear to be related to the risk of developing calcineurin inhibitor-related nephrotoxicity. However, the latter needs to be replicated before an individual's ABCB1 genotype can become a useful marker that is applied in clinical practice. Future studies evaluating the influence of ABC transporter gene polymorphisms should explore the relationship with intracellular rather than systemic drug concentrations further in well-designed clinical studies. Until then, single-nucleotide polymorphisms in ABC transporter genes are not suitable to act as biomarkers for solid organ transplantation.