Clinical performance of volumetric finger-prick sampling for the monitoring of tacrolimus, creatinine and haemoglobin in kidney transplant recipients.

AIMS: Finger-prick sampling has emerged as an attractive tool for therapeutic drug monitoring and associated diagnostics. We aimed to validate the clinical performance of using two volumetric devices (Capitainer® qDBS and Mitra®) for monitoring tacrolimus, creatinine and haemoglobin in kidney transplant (KTx) recipients. Secondarily, we evaluated potential differences between finger-prick sampling performed by healthcare professionals vs. self-sampling, and differences between the two devices. METHODS: We compared finger-prick and venous sampling in three settings: microsampling performed by healthcare personnel, self-sampling under supervision, unsupervised self-sampling. The finger-prick samples were analysed with adapted methods and results compared to routine method analysis of the venous blood samples. RESULTS: Twenty-five KTx recipients completed the main study and 12 KTx recipients completed a post hoc validation study. For tacrolimus measurements and predicted area under the curve, the proportions within ±20% difference were 79%-96% for Capitainer and 77%-95% for Mitra. For creatinine and haemoglobin, the proportions within ±15% were 92%-100% and 93%-100% for Capitainer and 79%-96% and 67%-92% for Mitra, respectively. Comparing sampling situations, the success rate was consistent for Capitainer (92%-96%), whereas Mitra showed 72%-88% and 52%-72% success rates with samples collected by healthcare personnel and the patients themselves. CONCLUSIONS: Capitainer and Mitra are technically feasible for measuring tacrolimus, creatinine and haemoglobin. In the context of self-sampling, Capitainer maintained consistent sampling success and analytical quality. Implementing volumetric finger-prick self-sampling for the monitoring of tacrolimus, creatinine and haemoglobin may simplify and improve the follow-up of KTx recipients.

Monitoring Simvastatin Adherence in Patients With Coronary Heart Disease: A Proof-of-Concept Study Based on Pharmacokinetic Measurements in Blood Plasma.

BACKGROUND: Poor statin adherence remains a public health concern associated with adverse outcomes. We evaluated the use of pharmacokinetic measurements to monitor adherence to simvastatin in patients with coronary heart disease (CHD). METHODS: Eighteen patients with CHD taking an evening dose of simvastatin 20 mg (n = 7), 40 mg (n = 5), or 80 mg (n = 6) were examined at steady-state pharmacokinetics. Ten patients were instructed to interrupt simvastatin dosing and return for blood sampling for the subsequent 3 days. Dose-normalized plasma concentrations of simvastatin lactone and simvastatin acid and the sum of the 2 were evaluated to discriminate between adherent dosing and dose omission. Bioanalytical quantification was performed using liquid chromatography-tandem mass spectrometry. RESULTS: A simvastatin acid cutoff of 1.0 × 10 -2 nmol -1 ·L -1 ·mg -1 identified 100% of those omitting 2 doses and 60% of those omitting a single dose. Simvastatin acid showed superior ability to discriminate dose omission, as well as the best agreement between samples handled at ambient and cool temperatures (median deviation 3.5%; interquartile range -2.5% to 13%). The cutoff for a morning dose schedule, with a similar ability to discriminate, was estimated at 2.0 × 10 -3 nmol -1 ·L -1 ·mg -1 . CONCLUSIONS: The present method discriminated between adherence and reduced adherence to simvastatin therapy in patients with CHD. Sample handling is feasible for routine practice, and the assessment of adherence can be performed by direct measurement of simvastatin acid in a blood sample, according to defined cutoff values. Further studies validating the cutoff value and utility for clinical application are encouraged.

Tacrolimus Measured in Capillary Volumetric Microsamples in Pediatric Patients-A Cross-Validation Study.

BACKGROUND: Therapeutic drug monitoring of tacrolimus (Tac) is mandatory in solid organ transplant (SOT) recipients. Finger-prick microsampling is more flexible and tolerable during the therapeutic drug monitoring of tacrolimus and has been shown to be applicable in adult SOT recipients. In this study, a previously validated method applying volumetric absorptive microsampling (VAMS) to measure Tac in adults was cross-validated in a pediatric population. METHODS: Patients with SOT scheduled for standard posttransplant follow-up visits were recruited. Blood samples were obtained by trained phlebotomists using standard venipuncture and capillary microsampling, before the morning dose of Tac as well as 2 and 5 hours after dosing. Tac concentrations were quantified using liquid chromatography-tandem mass spectrometry. Concordance between Tac concentrations obtained with venipuncture and VAMS was evaluated using Passing-Bablok regression, calculation of absolute and relative differences, and percentage of samples within ±20% and ±30% difference. RESULTS: A total of 39 SOT patients aged 4-18 years (22 male) were included. The median (range) predose venous blood concentration was 4.8 (2.6-13.6) mcg/L, with a difference between VAMS and venous blood samples of -0.2 ± 0.7 mcg/L. The relative mean difference was -1.3% [95% confidence interval (CI), -5.9% to 3.4%]. Ninety-two percent and 97% of the sample pairs demonstrated differences within ±20% and ±30%, respectively. Postdose (2 hours and/or 5 hours, n = 17) median concentration in venous blood was 7.9 (4.8-19.2) mcg/L. The difference between VAMS and venous blood samples was 0.1 ± 1.0 mcg/L, with a relative mean difference of -2.5% (95% confidence interval, -8.8% to 3.8%). Eighty-eight percent of the postdose sample pairs were within ±20% difference, and all were within ±30% difference. CONCLUSIONS: Tac concentrations can be accurately measured using VAMS technology in pediatric SOT recipients. This makes home-based Tac monitoring feasible in the pediatric population.

Personalized Therapy for Mycophenolate: Consensus Report by the International Association of Therapeutic Drug Monitoring and Clinical Toxicology.

ABSTRACT: When mycophenolic acid (MPA) was originally marketed for immunosuppressive therapy, fixed doses were recommended by the manufacturer. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA area under the curve based on the measurement of drug concentrations in only a few samples. This approach is feasible in the clinical routine and has proven successful in terms of correlation with outcome. However, the search for superior correlates has continued, and numerous studies in search of biomarkers that could better predict the perfect dosage for the individual patient have been published. As it was considered timely for an updated and comprehensive presentation of consensus on the status for personalized treatment with MPA, this report was prepared following an initiative from members of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT). Topics included are the criteria for analytics, methods to estimate exposure including pharmacometrics, the potential influence of pharmacogenetics, development of biomarkers, and the practical aspects of implementation of target concentration intervention. For selected topics with sufficient evidence, such as the application of limited sampling strategies for MPA area under the curve, graded recommendations on target ranges are presented. To provide a comprehensive review, this report also includes updates on the status of potential biomarkers including those which may be promising but with a low level of evidence. In view of the fact that there are very few new immunosuppressive drugs under development for the transplant field, it is likely that MPA will continue to be prescribed on a large scale in the upcoming years. Discontinuation of therapy due to adverse effects is relatively common, increasing the risk for late rejections, which may contribute to graft loss. Therefore, the continued search for innovative methods to better personalize MPA dosage is warranted.

Clinical and psychological factors in coronary heart disease patients with statin associated muscle side-effects.

BACKGROUND: To compare clinical and psychological factors among patients with self-perceived statin-associated muscle symptoms (SAMS), confirmed SAMS, and refuted SAMS in coronary heart disease patients (CHD). METHODS: Data were obtained from a cross-sectional study of 1100 CHD outpatients and a study of 71 CHD outpatients attending a randomized, double-blinded, placebo-controlled, crossover study to test effects of atorvastatin 40 mg/day on muscle symptom intensity. Clinical and psychosocial factors were compared between patients with and without SAMS in the cross-sectional study, and between patients with confirmed SAMS and refuted SAMS in the randomized study. RESULTS: Bilateral, symmetric muscle symptoms in the lower extremities during statin treatment were more prevalent in patients with confirmed SAMS compared to patients with refuted SAMS (75% vs. 41%, p = 0.01) in the randomized study. No significant differences in psychological factors (anxiety, depression, worry, insomnia, type D personality characteristics) were detected between patients with and without self-perceived SAMS in the cross-sectional study, or between patients with confirmed SAMS and refuted SAMS, in the randomized study. CONCLUSIONS: Patients with confirmed SAMS more often present with bilateral lower muscle symptoms compared to those with refuted SAMS. Psychological factors were not associated with self-perceived SAMS or confirmed SAMS. A careful pain history and a search for alternative causes of muscle symptoms are likely to promote communication in patients with SAMS, and may reduce the risk for statin discontinuation.

Pharmacodynamic assessment of mycophenolic acid in resting and activated target cell population during the first year after renal transplantation.

AIMS: To explore the pharmacodynamics of mycophenolic acid (MPA) through inosine monophosphate dehydrogenase (IMPDH) capacity measurement and purine levels in peripheral blood mononuclear cells (PBMC) longitudinally during the first year after renal transplantation (TX). METHODS: PBMC were isolated from renal recipients 0-4 days prior to and 6-9 days, 5-7 weeks and 1 year after TX (before and 1.5 hours after dose). IMPDH capacity and purine (guanine and adenine) levels were measured in stimulated and nonstimulated PBMC. RESULTS: Twenty-nine patients completed the follow-up period, of whom 24 received MPA. In stimulated PBMC, the IMPDH capacity (pmol 10-6 cells min-1 ) was median (interquartile range) 127 (95.8-147) before TX and thereafter 44.9 (19.2-93.2) predose and 12.1 (4.64-23.6) 1.5 hours postdose across study days after TX. The corresponding IMPDH capacity in nonstimulated PBMC was 5.71 (3.79-6.93), 3.35 (2.31-5.62) and 2.71 (1.38-4.08), respectively. Predose IMPDH capacity in nonstimulated PBMC increased with time, reaching pre-TX values at 1 year. In stimulated PBMC, both purines were reduced before (median 39% reduction across days after TX) and after (69% reduction) dose compared to before TX. No alteration in the purine levels was observed in nonstimulated PBMC. Patients needing dose reductions during the first year had lower pre-dose IMPDH capacity in nonstimulated PBMC (1.87 vs 3.00 pmol 10-6 cells min-1 , P = .049) at 6-9 days. CONCLUSION: The inhibitory effect of MPA was stronger in stimulated PBMC. Nonstimulated PBMC became less sensitive to MPA during the first year after TX. Early IMPDH capacity appeared to be predictive of dose reductions.

BEtablocker Treatment After acute Myocardial Infarction in revascularized patients without reduced left ventricular ejection fraction (BETAMI): Rationale and design of a prospective, randomized, open, blinded end point study.

BACKGROUND: Current guidelines on the use of ß-blockers in post-acute myocardial infarction (MI) patients without reduced left ventricular ejection fraction (LVEF) are based on studies before the implementation of modern reperfusion and secondary prevention therapies. It remains unknown whether ß-blockers will reduce mortality and recurrent MI in contemporary revascularized post-MI patients without reduced LVEF. DESIGN: BETAMI is a prospective, randomized, open, blinded end point multicenter study in 10,000 MI patients designed to test the superiority of oral ß-blocker therapy compared to no ß-blocker therapy. Patients with LVEF ≥40% following treatment with percutaneous coronary intervention or thrombolysis and/or no clinical signs of heart failure are eligible to participate. The primary end point is a composite of all-cause mortality or recurrent MI obtained from national registries over a mean follow-up period of 3 years. Safety end points include rates of nonfatal MI, all-cause mortality, ventricular arrhythmias, and hospitalizations for heart failure obtained from hospital medical records 30 days after randomization, and from national registries after 6 and 18 months. Key secondary end points include recurrent MI, heart failure, cardiovascular and all-cause mortality, and clinical outcomes linked to ß-blocker therapy including drug adherence, adverse effects, cardiovascular risk factors, psychosocial factors, and health economy. Statistical analyses will be conducted according to the intention-to-treat principle. A prespecified per-protocol analysis (patients truly on ß-blockers or not) will also be conducted. CONCLUSIONS: The results from the BETAMI trial may have the potential of changing current clinical practice for treatment with ß-blockers following MI in patients without reduced LVEF. EudraCT number 2018-000590-75.

A Method for Direct Monitoring of Atorvastatin Adherence in Cardiovascular Disease Prevention: Quantification of the Total Exposure to Parent Drug and Major Metabolites Using 2-Channel Chromatography and Tandem Mass Spectrometry.

BACKGROUND: Low adherence to statin therapy remains a public health concern associated with poor prognosis in cardiovascular disease patients. A feasible method for statin adherence monitoring in clinical practice has yet to be developed. In this article, we describe a novel method designed for the direct monitoring of atorvastatin adherence based on the sum of parent drug and major metabolites in blood samples. METHODS: Acid and lactone forms of atorvastatin, 2-OH-atorvastatin, and 4-OH-atorvastatin were assayed. Plasma proteins were precipitated with an acidified mixture of methanol, acetonitrile, and aqueous zinc sulfate, and the supernatant was analyzed with 2-channel reversed-phase chromatography coupled to tandem mass spectrometry. Assay validation was performed according to the guidelines provided by the European Medicines Agency and the US Food and Drug Administration. RESULTS: The effective run time was 1 minute and 45 seconds per sample. Mean accuracy ranged from 92% to 110%, and coefficients of variation were ≤8.1% over the measurement ranges for individual compounds. The sum of acids and corresponding lactones was stable in clinical plasma samples kept at ambient temperature for up to 6 days after blood sampling (mean sum within 96.6%-101% of baseline). CONCLUSIONS: A fast and reliable assay for the quantification of atorvastatin and its 5 major metabolites in clinical blood samples is reported. Limitations of preanalytical stability were solved using the sum of the acid and lactone forms. The assay is feasible for implementation in clinical practice, and the sum of parent drug and metabolites may be used for direct monitoring of atorvastatin adherence.

A Fully Automated Method for the Determination of Serum Belatacept and Its Application in a Pharmacokinetic Investigation in Renal Transplant Recipients.

BACKGROUND: Belatacept (Nulojix; Bristol-Myers Squibb, New York, NY) is a biological immunosuppressive drug used for the prophylaxis of acute rejection after renal transplantation. Few studies have described belatacept pharmacokinetics, and the effect of therapeutic drug monitoring has not been investigated. We have developed a drug-capture assay (using drug target) to measure belatacept in serum and applied this assay in a pharmacokinetic study in renal transplant recipients. METHODS: CD80 was used to trap belatacept onto streptavidin-coated wells. Captured drug was quantified using Eu-labeled protein A and time-resolved fluorescence. The assay was applied in a pilot pharmacokinetic study in renal transplanted patients receiving belatacept infusions. Belatacept serum concentrations were determined at several time points between belatacept infusions. A simple population pharmacokinetic model was developed to visualize measured and predicted belatacept serum concentrations. RESULTS: The assay range was 0.9-30 mg/L with accuracy within 91%-99% and coefficients of variation ranging from 1.2% to 3.6%. Predilution extended the measurement range to 130 mg/L with an accuracy of 90% and coefficients of variation of 3.8%. Samples were stable during storage at 4°C for 15 days and during 2 freeze-thaw cycles. Belatacept concentrations were determined in a total of 203 serum samples collected during 26 infusion intervals from 5 renal transplant recipients. The population pharmacokinetic model visualized both measured and predicted concentrations. CONCLUSIONS: We have developed an automated, accurate, and precise assay for the determination of belatacept serum concentrations. The assay was successfully applied in a pharmacokinetic study in renal transplant recipients receiving belatacept infusions.

Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report.

Ten years ago, a consensus report on the optimization of tacrolimus was published in this journal. In 2017, the Immunosuppressive Drugs Scientific Committee of the International Association of Therapeutic Drug Monitoring and Clinical Toxicity (IATDMCT) decided to issue an updated consensus report considering the most relevant advances in tacrolimus pharmacokinetics (PK), pharmacogenetics (PG), pharmacodynamics, and immunologic biomarkers, with the aim to provide analytical and drug-exposure recommendations to assist TDM professionals and clinicians to individualize tacrolimus TDM and treatment. The consensus is based on in-depth literature searches regarding each topic that is addressed in this document. Thirty-seven international experts in the field of TDM of tacrolimus as well as its PG and biomarkers contributed to the drafting of sections most relevant for their expertise. Whenever applicable, the quality of evidence and the strength of recommendations were graded according to a published grading guide. After iterated editing, the final version of the complete document was approved by all authors. For each category of solid organ and stem cell transplantation, the current state of PK monitoring is discussed and the specific targets of tacrolimus trough concentrations (predose sample C0) are presented for subgroups of patients along with the grading of these recommendations. In addition, tacrolimus area under the concentration-time curve determination is proposed as the best TDM option early after transplantation, at the time of immunosuppression minimization, for special populations, and specific clinical situations. For indications other than transplantation, the potentially effective tacrolimus concentrations in systemic treatment are discussed without formal grading. The importance of consistency, calibration, proficiency testing, and the requirement for standardization and need for traceability and reference materials is highlighted. The status for alternative approaches for tacrolimus TDM is presented including dried blood spots, volumetric absorptive microsampling, and the development of intracellular measurements of tacrolimus. The association between CYP3A5 genotype and tacrolimus dose requirement is consistent (Grading A I). So far, pharmacodynamic and immunologic biomarkers have not entered routine monitoring, but determination of residual nuclear factor of activated T cells-regulated gene expression supports the identification of renal transplant recipients at risk of rejection, infections, and malignancy (B II). In addition, monitoring intracellular T-cell IFN-g production can help to identify kidney and liver transplant recipients at high risk of acute rejection (B II) and select good candidates for immunosuppression minimization (B II). Although cell-free DNA seems a promising biomarker of acute donor injury and to assess the minimally effective C0 of tacrolimus, multicenter prospective interventional studies are required to better evaluate its clinical utility in solid organ transplantation. Population PK models including CYP3A5 and CYP3A4 genotypes will be considered to guide initial tacrolimus dosing. Future studies should investigate the clinical benefit of time-to-event models to better evaluate biomarkers as predictive of personal response, the risk of rejection, and graft outcome. The Expert Committee concludes that considerable advances in the different fields of tacrolimus monitoring have been achieved during this last decade. Continued efforts should focus on the opportunities to implement in clinical routine the combination of new standardized PK approaches with PG, and valid biomarkers to further personalize tacrolimus therapy and to improve long-term outcomes for treated patients.

Statin-associated muscle symptoms in coronary patients: design of a randomized study.

Objectives. Estimate the effect of atorvastatin on muscular symptom intensity in coronary patients with subjective statin-associated muscle symptoms (SAMS) and to determine the association with blood levels of atorvastatin and its metabolites, to obtain an objective marker for true SAMS. Design. A randomized, double-blinded, cross-over study will include 80 coronary patients with subjectively reported SAMS during ongoing atorvastatin therapy or previous muscle symptoms that led to discontinuation of atorvastatin. Patients will be randomized to 7-weeks treatment with atorvastatin 40 mg/day in the first period and matched placebo in the second 7-weeks period, or placebo in the first period and atorvastatin in the second period. Each period is preceded by 1-week wash-out. A control group (n = 40) without muscle symptoms will have 7 weeks open treatment with atorvastatin 40 mg/day. Blood samples will be collected at baseline and at the end of each treatment period, and muscular symptoms will be rated by the patients weekly using a Visual Analogue Scale (VAS). The primary outcome is the difference in aggregated mean VAS scores between the last three weeks of atorvastatin treatment and of placebo treatment. The main purpose is to develop an objective marker for true SAMS, by comparing SAMS associated with blinded atorvastatin treatment with blood concentrations of atorvastatin and its metabolites. Diagnostic and discrimination performance will be determined. Conclusions. The study provides new knowledge on SAMS in coronary patients and may contribute to more personalized statin treatment and monitoring, fewer side-effects and consequently improved adherence and lipid management in future practice.

Longitudinal Study of Tacrolimus in Lymphocytes During the First Year After Kidney Transplantation.

INTRODUCTION: Tacrolimus (TAC) is an immunosuppressive drug used after organ transplantation. Dosing is adjusted using whole blood (WB-TAC) measurements. Patients within the therapeutic WB-TAC window still experience rejections and adverse effects. Alternative monitoring methods are therefore warranted. The authors developed a method for measuring TAC in peripheral blood mononuclear cell (PBMC) isolates (PBMC-TAC) and performed a pharmacokinetic study in a cohort of kidney transplant patients during the first year after transplantation. METHODS: PBMCs were isolated from whole blood by gradient centrifugation. After methanol-based extraction, liquid chromatography with tandem mass spectrometry was used to determine TAC in the extract. PBMC-TAC was normalized to the number of cells and alternatively to the protein amount in cells. Predose and postdose (1.5 hours) samples from kidney transplant patients were collected at 1 week, 6 weeks, and 1 year after transplantation. WB-TAC was measured using immunoassay. RESULTS: The PBMC-TAC assay fulfilled the validation criteria of the European Medicines Agency guidelines. Twenty-nine patients completed the study. Predose PBMC-TAC was (median) 23 (1 week), 33 (6 weeks), and 27 pg/10 cells (1 year). Postdose PBMC-TAC was 44, 30, and 27 pg/10 cells at 1 week, 6 weeks, and 1 year after transplantation, respectively. Predose WB-TAC (median) was 5.0, 6.0, and 5.4 mcg/L, and postdose WB-TAC was 10.5, 8.3, and 9.1 mcg/L, respectively, at 1 week, 6 weeks, and 1 year after transplantation. Whole blood and PBMC-TAC correlated at all timepoints (rho 0.40-0.82, P < 0.05) except before dosage at 6 weeks. PBMC-TAC normalized to the number of cells, and the amount of protein was modestly correlated (rho 0.36-0.81, P < 0.056). CONCLUSIONS: The correlation between WB-TAC and PBMC-TAC is modest during the first-year posttransplantation. Normalization of PBMC-TAC to cells or protein may yield different results. PBMC-TAC is increased 1.5 hours after dose at 1 week after transplantation, but not after 6 weeks or 1 year, indicating altered distribution kinetics.

Prednisolone and Prednisone Pharmacokinetics in Pediatric Renal Transplant Recipients-A Prospective Study.

BACKGROUND: Prednisolone is a standard component of immunosuppressive protocols in renal transplantation (Tx) and despite standardized treatment regimens, adverse side effects are still frequent. The aim of this study was to characterize the pharmacokinetics of prednisolone and prednisone in pediatric renal transplant recipients in the first 52 weeks post Tx, to describe the relationship between prednisolone and prednisone, and to investigate a possible relationship between the development of new-onset diabetes after Tx (NODAT) and glucocorticoid exposure. METHODS: Renal transplant recipients receiving prednisolone (n = 11, age 1-15 years) were included in this prospective open-label, descriptive, nonrandomized, and noninterventional study. Blood samples were drawn pre-Tx and during selected dose intervals (0, 1, 2, 4, 6, and 12 hours postdose; less frequent in children <10 kg) at 1, 2, 3, 4, 12, and 52 weeks post-Tx. Concentrations of prednisolone and cortisol, their inactive keto forms, plus methylprednisolone, were measured using a validated LC-MS/MS method. Genetic variants in the CYP3A4, CYP3A5, ABCB1, and HSD11B2 genes were analyzed using real-time polymerase chain reaction and Sanger sequencing. Correlation with NODAT was investigated. RESULTS: The patients displayed considerable intra- and inter-individual variability in prednisolone exposure, with up to 5-fold differences in the area under the concentration-time curve (AUC). There were up to 7-fold differences in prednisolone/prednisone AUC ratio between patients, and patients experiencing NODAT tended to have a higher ratio (>12) compared with patients without NODAT (<12). Genetic variants in CYP3A5 and ABCB1 were found, but due to the limited study population causality cannot be definitive. CONCLUSIONS: The study suggests that a high prednisolone/prednisone AUC ratio may be a possible risk factor for NODAT. Further studies of individualization of glucocorticoid treatment in pediatric organ Tx are warranted.

Closer to the Site of Action: Everolimus Concentrations in Peripheral Blood Mononuclear Cells Correlate Well With Whole Blood Concentrations.

BACKGROUND: Everolimus (EVE) is an immunosuppressive drug dosed according to therapeutic drug monitoring in renal transplant recipients. The primary site of action is within activated lymphocytes. EVE is a substrate of the efflux transporter ABCB1 also known as P-glycoprotein. Limited data exist regarding a possible association between whole blood and intralymphocyte concentrations of EVE and the potential influence of ABCB1. METHODS: Twelve renal transplant recipients (5 men and 7 women) treated with EVE underwent a pharmacokinetic investigation where EVE concentrations in whole blood and in peripheral blood mononuclear cells (PBMC) were determined within a dosing interval. In addition, the activity of ABCB1 in PBMC was determined using the Rhodamine123 efflux assay and the patients' genotypes of ABCB1 were determined. RESULTS: There was a significant correlation between EVE AUC0-12 in whole blood and in PBMC (r = 0.90, P < 0.01), and no association was demonstrated between the ABCB1 activity and EVE PBMC/whole blood ratio of trough concentrations (r = 0.23, P = 0.76). Furthermore, ABCB1 1236C>T, 3435C>T, and 2677G>T/A polymorphism did not influence EVE AUC0-12 PBMC/whole blood ratio. CONCLUSIONS: The results revealed a significant association between EVE whole blood and PBMC concentrations, suggesting that ABCB1-mediated efflux from PBMC to be of minor importance for the distribution of EVE.

Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

MAIN PROBLEM: Islet transplantation has become a promising treatment for type 1 diabetes. However, immunosuppressive drugs used today cause islet deterioration and modification strategies are necessary. But little is known about pharmacokinetics interactions and intracellular concentrations of immunosuppressive drugs in human islets. METHODS: We determined the pharmacokinetics of tacrolimus and sirolimus in islets by measuring intracellular concentration after exposure alone or in combination at two different doses up to 48 h. A quantification technique established in our laboratory using a Micromass Quattro micro API MS/MS-instrument with electrospray ionization was used. Islets function was measured by oxygen consumption rates. Presence of drug transporters OATP1B1 and ABCB1 and metabolizing enzyme CYP3A4 in islets were quantified using real-time quantitative PCR. RESULTS: Islets incubated with tacrolimus and sirolimus had a significant decrease in intracellular concentration of sirolimus compared to sirolimus alone. Reduced intracellular sirolimus concentration was followed by increased p70S6k phosphorylation suggesting preservation of the mTOR-signaling pathway. Drug transporters OATP1B1 and ABCB1 and enzyme CYP3A4 were expressed in human islets, but were not involved in the reduced sirolimus concentration by tacrolimus. CONCLUSION: These findings provide new knowledge of the drug interaction between tacrolimus and sirolimus, suggesting that tacrolimus has an inhibitory effect on the intracellular concentration of sirolimus in human islets.

Simultaneous quantification of IMPDH activity and purine bases in lymphocytes using LC-MS/MS: assessment of biomarker responses to mycophenolic acid.

BACKGROUND: The development of biomarkers describing the individual responses to the immunosuppressant mycophenolic acid (MPA) has focused on the target enzyme activity [inosine 5'-monophosphate dehydrogenase (IMPDH)]. An extended strategy is to quantify the metabolic consequences of IMPDH inhibition. The aim of this study was to develop an assay for quantification of IMPDH activity and related purine bases and to provide preliminary data on the behavior of these biomarkers during clinical exposure to MPA. METHODS: Liquid chromatography-mass spectrometry was used to determine xanthine (IMPDH activity in incubated cell lysate), hypoxanthine, guanine, and adenine derived from free nucleotides in lymphocytes. Analytical performance was assessed, and the biomarkers were examined in CD4⁺ cells from 2 groups: Healthy individuals in a single-dose MPA study (n = 5) and liver transplant recipients on MPA therapy (n = 15). RESULTS: Coefficients of variation between series were below 10% and 15% for measurement of the purines and IMPDH activity, respectively. Although IMPDH was inhibited, the purine levels increased in response to MPA in 3 of the 5 healthy individuals, and this positive response seemed to be associated with IMPDH1 c.579 + 119 G/G and c.580 - 106 G/G. In the liver transplant study, guanine was not reduced in response to the transient drop in IMPDH activity after MPA dosing. However, there were trends toward decrease in guanine and elevation of hypoxanthine during prolonged MPA therapy. The guanine/hypoxanthine ratio (median) was 37% lower and the adenine level was 21% lower at day 17 compared with day 4 after transplantation. CONCLUSIONS: The assay allows precise quantification of IMPDH activity, hypoxanthine, guanine, and adenine in lymphocytes. Some individuals may possess a counteracting purine response to the MPA-mediated inhibition of IMPDH. Reduction of the guanine/hypoxanthine ratio may be related to prolonged inhibition of IMPDH and seems as an intriguing pharmacodynamic biomarker for MPA.