Monitoring of tacrolimus concentrations in peripheral blood mononuclear cells: Application to cardiac transplant recipients

doi:10.1016/j.clinbiochem.2013.02.011

Clinical Biochemistry

Volume 46, Issue 15, October 2013, Pages 1538-1541

https://doi.org/10.1016/j.clinbiochem.2013.02.011 Get rights and content

Abstract

Objectives

Despite the intensive therapeutic drug monitoring of tacrolimus (TAC) using trough whole blood concentrations, graft rejections occur in transplant recipient patients. Thus, other ways to monitor closely immunosuppressive treatments are necessary. A promising way of monitoring TAC treatments could be the measure of its concentrations inside of the lymphocyte cell. Whereas the pharmacokinetics of TAC in peripheral blood mononuclear cells (PBMCs) was evaluated in renal and liver transplant recipients, data regarding PBMC concentrations of TAC in cardiac transplant recipients are lacking.

This study aimed, in cardiac transplant recipients: to validate a method for determination of TAC in PBMC, to investigate PBMC trough concentrations of TAC, and to evaluate their relationship with trough whole blood concentrations.

Design and method

We developped and validated a High-performance-liquid-chromatography tandem mass-spectrometry method of TAC quantitation in PBMC. The method was then evaluated by determining TAC concentrations in PBMC of 24 cardiac transplant recipients.

Results

Twenty-four patients were prospectively included in the study.

Tacrolimus PBMC concentrations displayed a large inter-individual pharmacokinetic variability (CV = 71.4%) in the cohort. A lack of correlation between TAC whole blood trough concentrations and TAC trough concentrations in PBMCs was found (r = 0.259; p = 0.183).

Conclusion

Further studies should be implemented to evaluate the correlation between TAC concentrations in PBMC and clinical outcomes in cardiac transplant recipients to allow concluding whether monitoring TAC concentrations in PBMC is a good tool to prevent graft rejection in cardiac recipients.

Highlights

► Determination of tacrolimus in peripheral blood mononuclear cells is proposed. ► First application of tacrolimus determination in PBMC in cardiac recipients. ► No correlation was found between TAC concentrations in PBMC and in whole blood. ► Large inter-individual variability of TAC concentrations in PBMC was highlighted. ► Monitoring TAC in PBMC could be more relevant to assess efficacy than in blood.

Introduction

Tacrolimus (TAC) is an immunosuppressive agent which is a pivotal treatment in several types of solid transplantations including cardiac, renal, hepatic, pulmonary and bone-marrow transplantations. Its narrow therapeutic index and the large inter- and intra-individual variability described in TAC pharmacokinetics led to a recommendation for a therapeutic drug monitoring (TDM) of whole blood trough concentrations (C0) in patients treated [1], [2]. Causes of variability were extensively studied and involved variations in absorption, distribution, metabolism and elimination pharmacokinetic phases [1].

Furthermore, TAC is a substrate of P-glycoprotein (ABCB1) and cytochrome P450 3A5 (CYP3A5) [2]. Thus, pharmacogenetics has also to be considered. The polymorphism of CYP3A5 and ABCB1 genes could lead to variability in TAC pharmacokinetics by increasing or decreasing TAC absorption or metabolism. A target level C0 between 5 and 10 ng/mL is currently proposed after the post-operative acute phase but data from the SYMPHONY study and a recent European consortium suggested a decreased target range (3–7 ng/mL) in renal recipients treated with mycophenolate mofetil plus corticosteroids or m-TOR inhibitor [3], [4].

Despite this intensive TDM, graft rejections occur and early acute rejection rates remain between 8 and 15% [4]. Graft rejections also occur when trough concentrations are inside therapeutic range. The current TDM approaches are limited in their ability to measure drug effectiveness at its immunosuppressive site of action. Thus, other ways to monitor immunosuppressive treatments could improve patient management.

The immunosuppressive action of calcineurin inhibitors exerts inside of the T-lymphocyte cell where TAC binds the FK-binding protein 12 (FKBP12). The complex TAC-FKBP12 inhibits then the calcineurin, a calcium-dependent protein involved in the first phase of T-cell activation [5].

Hence, the measurement of whole-blood or extracellular blood concentrations does not necessarily provide an accurate and reproducible reflect of intracellular drug concentrations within T cells.

Falck et al. reported a decrease in intra-lymphocyte concentrations of cyclosporine in kidney transplant recipients a few days before an episode of acute rejection whereas the whole blood concentrations of cyclosporine remain within therapeutic ranges. Hence, intra-lymphocyte concentrations of cyclosporine could be a more predictive sign of acute rejection [6].

As the TAC immunosuppressive effect is also mediated through the inhibition of calcineurin in lymphocytes, direct quantification within this target compartment would appear to have a closer association with drug efficacy than whole blood concentrations or even tissue concentrations. Lymphocyte concentrations could provide more consistent information.

Drug-efflux transporters such as P-glycoprotein (P-gp) could significantly affect the distribution of immunosuppressive drugs in blood cells. This efflux pump is expressed on lymphocyte and monocyte membranes. Polymorphism of ATP binding cassette could lead to a potentially high variability of the TAC pharmacokinetics in peripheral blood mononuclear cells (PBMCs) [7].

A liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been used to quantify TAC in PBMC and experienced in kidney transplant or liver transplant recipients [8], [9]. No data regarding PBMC concentrations of TAC in cardiac transplant recipients has been described in literature.

The aim of this study was to validate a method for the determination of TAC in PBMC and to investigate the relation between intracellular and trough whole blood concentrations of TAC, physiological and biological parameters in cardiac transplant recipients.

Section snippets

Patients and data collection

Twenty-four patients treated with TAC (19 with immediate release formulation, 5 with modified release formulation) for the prevention of cardiac graft rejection were prospectively included in this study from the 15th August 2011 to the 31st November 2011 and PBMC concentrations of TAC were determined in 28 samples. No supplementary blood samples were drawn and investigations were considered part of routine clinical practice. Nevertheless, patients were informed of their inclusion in the study

Method validation

The present LC–MS/MS method was validated according to the ICH criteria. Calibration curves for TAC were linear over a range of 12.5–250 pg/million PBMCs over the three days of experiments with correlation coefficient values ranging from 0.984 to 0.997.

The intra-day coefficients of variation for TAC controls (50 and 200 pg/million PBMCs) were respectively 8.6 and 9.0%. Coefficients of variation of inter-day analysis of TAC controls (50 and 200 pg/million PBMC) were respectively 2.8 and 8.8%. Data

Discussion

We have reported here the first study evaluating the pharmacokinetics of TAC in PBMC in cardiac transplant recipients. We have presented a fully validated LC–MS/MS method to evaluate TAC concentrations in PBMCs. The linearity of the method allows determination of TAC in PBMCs in patients on the early post-operative period despite the low number of T cells, as well as in patients treated for a long time. Accuracy and precision were satisfactory. Our results confirmed, in cardiac transplant

Conclusion

This study reports the first investigation of TAC pharmacokinetics in PBMC in cardiac transplant recipients. No biological or physiological parameter correlated with TAC concentrations in PBMC except creatininemia. A lack of correlation between TAC dosage, TAC whole blood trough concentrations and TAC trough concentrations in PBMC was found (r = 0.259; p = 0.183) confirming, in cardiac transplant recipients, the results previously published in renal and liver transplant recipients. Tacrolimus

Authors' contributions

Florian Lemaitre: Concept/design, pharmacokinetic and data analysis, statistics, and drafting article.

Marie Antignac: Pharmacokinetic analysis and statistics.

Christine Fernandez: Concept/design, critical revision of article and approval of article.

Acknowledgment

The authors would like to thank Dr Catherine Settegrana and her staff for their technical help with this work.

References (12)

M. Oellerich et al.
Therapeutic drug monitoring of cyclosporine and tacrolimus. Update on Lake Louise Consensus Conference on cyclosporin and tacrolimus
Clin Biochem
(1998)
F. Roullet-Renoleau et al.
Everolimus quantification in peripheral blood mononuclear cells using ultra high performance liquid chromatography tandem mass spectrometry
J Pharm Biomed Anal
(2012)
C.E. Staatz et al.
Clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ transplantation
Clin Pharmacokinet
(2004)
H. Ekberg et al.
Reduced exposure to calcineurin inhibitors in renal transplantation
N Engl J Med
(2007)
P. Wallemacq et al.
Opportunities to optimize tacrolimus therapy in solid organ transplantation: report of the European Consensus Conference
Ther Drug Monit
(2009)
P.F. Halloran
Immunosuppressive drugs for kidney transplantation
N Engl J Med
(2004)

There are more references available in the full text version of this article.

Cited by (50)

Intracellular tacrolimus concentration correlates with impaired renal function through regulation of the IS-AHR-ABC transporter in peripheral blood mononuclear cells
2024, International Immunopharmacology
Tacrolimus (TAC) concentration in peripheral blood mononuclear cells (PBMCs) is regarded as a better predictor of its immunosuppressive effect than the TAC concentration in whole blood. However, whether the exposure of TAC in PBMCs or WB was altered in post-transplant recipients with renal impairment remains unclear.
We investigated the relationship of trough TAC concentration in WB and PBMCs with renal functions in post-transplant recipients. The pharmacokinetic profiles of TAC in PBMCs and WB in the two chronic kidney disease (CKD) rat models were examined using UPLC-MS/MS. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to analyze the expression of proteins and mRNAs related to TAC metabolism and transport, respectively. In addition, the effects of uremic toxins on human PBMCs were investigated using whole-transcriptome sequencing (RNA sequencing [RNA-seq]).
We observed a decrease in the trough TAC concentration in PBMCs in the recipients with estimated glomerular filtration rate (eGFR) < 90 mL/min, compared with those of recipients with eGFR > 90 mL/min, but there was no difference in blood based on TAC concentrations (C_0Blood). In a 150-patient post-transplant cohort, no significant relationship was observed between PBMCs and WB concentrations of TAC, and the eGFR value was correlated with TAC C_0PBMCs but not with TAC C_0Blood. In two CKD rat models, the TAC pharmacokinetic profile in the PBMCs was significantly lower than that in the control group; however, the blood TAC pharmacokinetic profiles in the two groups were similar. Transcriptome results showed that co-incubation of human PBMCs with uremic toxins upregulated the expression of AHR, ABCB1, and ABCC2. Compared to control rats, plasma IS increased by 1.93- and 2.26-fold and the expression of AHR, P-gp, and MRP2 in PBMCs was higher in AD and 5/6 nephrectomy (NX) rats, without modifying the expression of other proteins related to TAC exposure.
The pharmacokinetics of TAC in PBMCs changed with a decline in renal function. Uremic toxins accumulate during renal insufficiency, which activates AHR, upregulates the expression of P-gp and MRP2, and affects their intracellular concentrations. Our findings suggest that monitoring TAC concentrations in PBMCs is more important than monitoring WB concentrations in post-transplant recipients with renal impairment.
A highly sensitive method for determination of tacrolimus in peripheral blood mononuclear cells by nano liquid chromatography–high resolution accurate mass spectrometry
2023, Journal of Chromatography A
The determination of intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) is crucial for assessing the effect-site concentration of tacrolimus. Analytical methods previously reported required a minimum of 3 mL of whole blood sample for measuring the tacrolimus concentration. In this study, we developed a highly sensitive method using EASY nLC 1200 combined with Q Exactive orbitrap mass spectrometer for detecting tacrolimus in PBMCs, requiring only 0.5–2 mL of sample. Furthermore, we compared two primary normalization methods for PBMCs tacrolimus concentration using Passing–Bablok regression, Bland–Altman analysis, Spearman's rank correlation, and Mountain plot. The newly established method was employed to compare tacrolimus concentrations in whole blood and PBMCs among 194 lung transplant recipients. The developed method exhibited high sensitivity with a lower limit of quantitation at 5 pg/mL, and excellent intra- and inter-days accuracy and precision. The comparison between different normalization methods for PBMCs tacrolimus concentration revealed a strong correlation between PBMCs count and intracellular protein amount within these cells. This finding suggests that both PBMCs count and intracellular protein amount can be used for normalizing intracellular tacrolimus levels and can be mutually converted. However, a weaker correlation was observed between PBMCs and whole-blood tacrolimus concentrations in lung transplant recipients, warranting further investigation. The method reported herein enables the quantification of PBMCs tacrolimus concentration using smaller volumes of whole blood samples, which has significant implications for both patients and laboratory personnel.
Expeditious quantification of plasma tacrolimus with liquid chromatography tandem mass spectrometry in solid organ transplantation
2023, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Traditionally, tacrolimus is assessed in whole blood samples, but this is suboptimal from the perspective that erythrocyte-bound tacrolimus is not a good representative of the active fraction. In this work, a straightforward and rapid method was developed for determination of plasma tacrolimus in solid organ transplant recipients, using liquid chromatography tandem mass spectrometry (LC-MS/MS) with heated electrospray ionisation. Sample preparation was performed through protein precipitation of 200 µl plasma with 500 µl stable isotopically labelled tacrolimus I.S. in methanol, where 20 µl was injected on the LC-MS/MS system. Separation was done using a chromatographic gradient on a C18 column (50 × 2.1 mm, 2.6 µm). The method was linear in the concentration range 0.05–5.00 µg/L, with within-run and between-run precision in the range 2–6 % and a run time of 1.5 min. Furthermore, the method was validated for selectivity, sensitivity, carry-over, accuracy and precision, process efficiency, recovery, matrix effect, and stability following EMA and FDA guidelines. Clinical validation was performed in 2333 samples from 1325 solid organ transplant recipients using tacrolimus (liver n = 312, kidney n = 1714, and lung n = 307), which had median plasma tacrolimus trough concentrations of 0.10 µg/L, 0.15 µg/L and 0.23 µg/L, respectively. This method is suitable for measurement of tacrolimus in plasma and will facilitate ongoing observational and prospective studies on the relationship of plasma tacrolimus concentrations with clinical outcomes.
Factors Affecting Day-to-Day Variations in Tacrolimus Concentration among Children and Young Adults Undergoing Allogeneic Hematopoietic Stem Cell Transplantation
2023, Transplantation and Cellular Therapy
Tacrolimus is widely used as prophylaxis for graft-versus-host disease (GVHD) in allogeneic stem cell transplantation (allo-HSCT). It has a narrow therapeutic index range; high tacrolimus concentrations are associated with toxicity, whereas low concentrations are associated with an increased risk of GVHD. Although dose adjustments based on therapeutic drug monitoring are performed, unexpected large variations in tacrolimus concentration are sometimes encountered. The available evidence suggests that the factors affecting tacrolimus concentration are not fully understood. This study was aimed primarily at investigating the factors affecting day-to-day variations in tacrolimus concentration in children and young adults who received continuous tacrolimus infusion after allo-HSCT. The secondary objective was to identify the factors causing large variations (>20%) in tacrolimus concentrations. This retrospective cohort study comprised 123 consecutive pediatric and young adult patients (age <25 years) who received continuous i.v. tacrolimus infusion after allo-HSCT at Shinshu University Hospital, Matsumoto, Japan, between January 2009 and December 2021. To compare day-to-day variations in tacrolimus concentration without consideration of the tacrolimus dose, 2 consecutive days when the tacrolimus dose was not changed were selected from between the first post-allo-HSCT day of a tacrolimus concentration >7 ng/mL and day 28 post-allo-HSCT. Subsequently, information for the subsequent 24 hours was collected along with the tacrolimus concentrations and hematocrit values. Tacrolimus concentration was determined using whole blood samples. Tacrolimus concentrations were significantly higher in patients who received red blood cell concentrate (RCC) transfusions (P < .0001) and methotrexate (P = .0162), patients with persistent fever (P = .0056), and patients with a decline in fever (P = .0003). In contrast, tacrolimus concentrations were significantly lower in patients who received platelet concentrate (PC) transfusions (P < .0001), who redeveloped fever (P = .0261), and who had a replaced tacrolimus administration route set (P = .0008). Variations in tacrolimus concentration were significantly correlated with variations in hematocrit (r = .556; P < .0001). Body weight (P < .0001), RCC transfusion (P < .0001), methotrexate use (P = .0333), persistent fever (P = .0150), and decline in fever (P = .0073) were associated with a sharp increase in tacrolimus concentration. In contrast, body weight (P < .0001), PC transfusion (P = .0025), and replacement of the tacrolimus administration route set (P = .0025) were associated with a sharp decrease in tacrolimus concentration. RCC and PC transfusions, fever, methotrexate administration, and replacement of the tacrolimus administration route set were independent factors affecting day-to-day variations in tacrolimus concentration. In addition to these factors, low body weight was a risk factor for both sharp increases and decreases in tacrolimus concentration. These findings suggest the need for better control of tacrolimus concentration using whole blood samples.
Drug transporters are implicated in the diffusion of tacrolimus into the T lymphocyte in kidney and liver transplant recipients: Genetic, mRNA, protein expression, and functionality
2022, Drug Metabolism and Pharmacokinetics
Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (C_blood) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (C_PBMC) could improve patient outcomes. The poor correlation between C_blood and C_PBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC C_PBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC C_PBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the C_PBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane.
Monitoring intracellular tacrolimus concentrations and its relationship with rejection in the early phase after renal transplantation
2022, Clinical Biochemistry
After kidney transplantation, rejection and drug-related toxicity occur despite tacrolimus whole-blood pre-dose concentrations ([Tac]_blood) being within the target range. The tacrolimus concentration within peripheral blood mononuclear cells ([Tac]_cells) might correlate better with clinical outcomes. The aim of this study was to investigate the correlation between [Tac]_blood and [Tac]_cells, the evolution of [Tac]_cells and the [Tac]_cells/[Tac]_blood ratio, and to assess the relationship between tacrolimus concentrations and the occurrence of rejection.
In this prospective study, samples for the measurement of [Tac]_blood and [Tac]_cells were collected on days 3 and 10 after kidney transplantation, and on the morning of a for-cause kidney transplant biopsy. Biopsies were reviewed according to the Banff 2019 update.
Eighty-three [Tac]_cells samples were measured of 44 kidney transplant recipients. The correlation between [Tac]_cells and [Tac]_blood was poor (Pearson’s r = 0.56 (day 3); r = 0.20 (day 10)). Both the dose-corrected [Tac]_cells and the [Tac]_cells/[Tac]_blood ratio were not significantly different between days 3 and 10, and the median inter-occasion variability of the dose-corrected [Tac]_cells and the [Tac]_cells/[Tac]_blood ratio were 19.4% and 23.4%, respectively (n = 24). Neither [Tac]_cells, [Tac]_blood, nor the [Tac]_cells/[Tac]_blood ratio were significantly different between patients with biopsy-proven acute rejection (n = 4) and patients with acute tubular necrosis (n = 4) or a cancelled biopsy (n = 9; p > 0.05).
Tacrolimus exposure and distribution appeared stable in the early phase after transplantation. [Tac]_cells was not significantly associated with the occurrence of rejection. A possible explanation for these results might be related to the low number of patients included in this study and also due to the fact that PBMCs are not a specific enough matrix to monitor tacrolimus concentrations.

View all citing articles on Scopus

View full text

Monitoring of tacrolimus concentrations in peripheral blood mononuclear cells: Application to cardiac transplant recipients

Abstract

Objectives

Design and method

Results

Conclusion

Highlights

Introduction

Section snippets

Patients and data collection

Method validation

Discussion

Conclusion

Authors' contributions

Acknowledgment

Clin Biochem

J Pharm Biomed Anal

Clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ transplantation

Clin Pharmacokinet

Reduced exposure to calcineurin inhibitors in renal transplantation

N Engl J Med

Opportunities to optimize tacrolimus therapy in solid organ transplantation: report of the European Consensus Conference

Ther Drug Monit

Immunosuppressive drugs for kidney transplantation

N Engl J Med