Aims
Despite pharmacokinetic monitoring of calcineurin inhibitors, the long‐term outcome after transplantation (Tx) is still hampered by the side effects of these drugs. The aim of the present study ...was to characterize nuclear factor of activated T cells (NFAT)‐regulated gene expression as a potential pharmacodynamic biomarker for further individualization of tacrolimus (Tac) therapy.
Methods
In 29 renal allograft recipients, samples were drawn once pre‐Tx, and before and 1.5 h after Tac dosing at approximately 1 week, 6 weeks and 1 year post‐Tx. Tac concentrations were measured by immunoassay, while the expression of genes encoding NFAT‐regulated cytokines interleukin 2 (IL2), interferon gamma (IFNG), colony stimulating factor 2 (CSF2) and cytochrome P450 3A5 (CYP3A5) genotyping were determined by real‐time polymerase chain reaction.
Results
The cytokine response after Tac dosing varied up to 46‐fold between patients and changed significantly with time post‐engraftment. Tac concentrations 1.5 h postdose (C1.5) >15 μg l–1 were associated with strong cytokine inhibition and residual gene expression (RGE) ≤10%, while lower Tac C1.5 resulted in more variable responses (RGE 2.5–68.7%). Patients with ongoing subclinical acute rejection (n = 5) demonstrated limited cytokine inhibition (RGE 39.7–72.6%), while patients with polyoma virus viraemia (n = 3) had relatively strong inhibition of cytokines (RGE 2.5–32.5%). By contrast, there was no association between Tac exposure and rejection or viraemia.
Conclusions
The findings of our study support the potential of NFAT‐regulated gene expression measurements as a pharmacodynamic tool for additional monitoring of Tac therapy, especially in the context of overimmunosuppression and viraemia.
Aims
Objective methods to monitor statin adherence are needed. We have established a liquid chromatography–tandem mass spectrometry assay for quantification of atorvastatin and its metabolites in ...blood. This study aimed to develop an objective drug exposure variable with cut‐off values to discriminate among adherence, partial adherence and nonadherence to atorvastatin therapy in patients with coronary heart disease.
Methods
Twenty‐five patients treated with atorvastatin 10 mg (n = 5), 20 mg (n = 6), 40 mg (n = 7) and 80 mg (n = 7) participated in a directly observed atorvastatin therapy study to confirm baseline adherence. After the directly observed therapy, half of the patients (test group) were instructed to stop taking atorvastatin and return for blood sample collection the subsequent 3 days. Levels of atorvastatin and metabolites were compared between the test group and the adherent control group.
Results
The sum of parent drug and all measured primary metabolites correlated well with the atorvastatin dose administered (Spearman's rho = 0.71, 95% CI 0.44–0.87). The dose‐normalized atorvastatin plus metabolites concentrations completely separated the partially adherent test group from the controls at 0.18 nM/mg after 3 days without atorvastatin. To reduce the risk of misinterpreting adherent patients as partially adherent, a corresponding cut‐off at 0.10 nM/mg is proposed. A metabolite level of 2‐OH atorvastatin acid <0.014 nmol/L provided the optimal cut‐off for nonadherence.
Conclusion
A direct method to discriminate among adherence, partial adherence and nonadherence to atorvastatin therapy in patients with coronary heart disease has been developed. This tool may be important for novel studies on adherence and potentially useful in clinical practice.
Therapeutic drug monitoring is standard practice for the immunosuppressant tacrolimus (Tac). Venous blood sampling at outpatient clinics is time-consuming and impractical with regard to obtaining ...trough concentrations on clinical visit days. Home-based blood sampling may be patient friendly and pave the way for limited sampling strategies for the prediction of total drug exposure. The aim was to establish a Tac assay for dried capillary microsamples, ensuring reliable measurements during the full dose interval in renal transplant recipients.
An assay based on volumetric absorptive microsampling and liquid chromatography tandem mass spectrometry was validated. The agreement between capillary microsamples and liquid venous samples was investigated in stable renal recipients on twice-daily Tac dosing. Sampling throughout the 12-hour dose interval was examined at 2 separate days, at least 1 week apart, for each participant. Two sets of samples were obtained at each time point, one delivered directly to the laboratory and one sent through mail.
Twenty-seven renal transplant recipients were included, of whom 26 were investigated twice. Tac was efficiently extracted from the dried microsamples (mean recovery 94%-103%). The between-series mean accuracy was 88%-98% with coefficients of variation ≤5.0% (≤11% at the lower limit of quantification), measurement range 0.70-60 mcg/L. The mean difference between parallel microsamples was 5%-7%. Overall, the mean differences between dried microsamples and liquid samples were -3.1% when mailed (n = 679) and -4.2% when directly delivered (n = 682). Less than 8% were outside ±20%. The microsamples were stable for 1 month at ambient temperature.
The microsample method demonstrated acceptable performance. Tac concentrations can be reliably quantified throughout the dose interval by using volumetric absorptive microsampling in renal transplant recipients, and the results are not influenced by postal shipment.
Therapeutic drug monitoring (TDM) of tacrolimus (Tac) is mandatory in renal transplant recipients (RTxR). Area under the concentration versus time curve (AUC) is the preferred measure for Tac ...exposure; however, for practical purposes, most centers use trough concentrations as a clinical surrogate. Limited sampling strategies in combination with population pharmacokinetic model-derived Bayesian estimators (popPK-BE) may accurately predict individual AUC. The use of self-collected capillary microsamples could simplify this strategy. This study aimed to investigate the potential of AUC-targeted Tac TDM using capillary microsamples in combination with popPK-BE.
A single-center prospective pharmacokinetic study was conducted in standard-risk RTxR (n = 27) receiving Tac twice daily. Both venous and capillary microsamples (Mitra; Neoteryx, Torrance, CA) were obtained across 2 separate 12-hour Tac dosing intervals (n = 13 samples/AUC). Using popPK-BE, reference AUC (AUCref) was determined for each patient using all venous samples. Different limited sampling strategies were tested for AUC predictions: (1) the empiric sampling scheme; 0, 1, and 3 hours after dose and (2) 3 sampling times determined by the multiple model optimal sampling time function in Pmetrics. Agreement between the predicted AUCs and AUCref were evaluated using C-statistics. Accepted agreement was defined as a total deviation index ≤±15%.
The AUC from capillary microsamples revealed high accuracy and precision compared with venous AUCref, and 85% of the AUCs had an error within ±11.9%. Applying microsamples at 0, 1, and 3 hours after dose predicted venous AUCref with acceptable agreement. Patients performed self-sampling with acceptable accuracy.
Capillary microsampling is patient-centered, making AUC-targeted TDM of Tac feasible without extended hospital stays. Samples obtained 0, 1, and 3 hours after dose, combined with popPK-BE, accurately predict venous Tac AUC.
Prednisolone (PL) is a standard component of most immunosuppressive protocols after solid organ transplantation (Tx). Adverse effects are frequent and well known. The aim of this study was to ...characterize the pharmacokinetics (PKs) of PL and prednisone (PN), including cortisol (CL) and cortisone (CN) profiles, after PL treatment in renal Tx recipients in the early post-Tx phase.
This single-center, prospective, observational study included stable renal Tx recipients, >18 years of age, and in the early postengraftment phase. Blood samples were obtained predose and during a 24-hour dose interval n = 26 samples per area under the curve (AUC0-24), within the first 8 weeks post-Tx. PL, PN, CL, and CN concentrations were measured using high-performance liquid chromatography-tandem mass spectrometry.
In renal Tx recipients (n = 28), our results indicated a relatively high PL exposure median, range AUC0-24 = 3821 (2232-5382) mcg h/L, paralleled by strong suppression of endogenous CL profile, demonstrated by a low CL evening-to-morning ratio median, range 11 (3-47)%. A negative correlation (r = -0.83) between PL AUC0-24 and morning CL levels was observed. The best single PK variable to predict PL AUC0-24 was PL C6 (r2 = 0.82). An algorithm based on 3 PK sampling time points: trough, 2, and 4 hours after PL dosing, predicted PL AUC0-24 with a low percentage prediction error (PPE = 5.2 ± 1.5%) and a good correlation of determination (r2 = 0.91). PL AUC0-24 varied 3-fold among study participants, whereas CL AUC0-24 varied by 18-fold.
The large interindividual variability in both PL exposure and suppression of endogenous CL implies a possible role for therapeutic drug monitoring. An abbreviated profile within the first 4 hours after PL dosing provides a good prediction of PL exposure in renal Tx recipients. The strong negative correlation between PL AUC0-24 and morning CL levels suggests a possible surrogate marker for drug exposure for further evaluation.
Tacrolimus (TAC) is currently the cornerstone of immunosuppressive protocols for renal transplant recipients. Despite therapeutic whole blood monitoring, TAC is associated with nephrotoxicity, and it ...has been hypothesized that intrarenal accumulation of TAC and/or its metabolites are involved. As TAC is a substrate of P-glycoprotein (P-gp), the expression and activity of this efflux transporter could influence the levels of TAC in renal tissue. The primary aim of this study was to develop and validate a method for quantification of TAC in tissue homogenates from single human renal core biopsies. The secondary aim was to provide measures of P-gp expression and of the demethylated metabolites of TAC in the same renal biopsy.
Human renal tissue, with and without clinical TAC exposure, was used for method development and validation. Homogenates were prepared with bead-beating, and concentrations of TAC and its demethylated metabolites were analyzed with liquid chromatography tandem mass spectrometry after protein precipitation. A Western blot method was used for semiquantification of P-gp expression in the homogenates. The final methods were applied to renal core biopsies from 2 transplant patients.
The TAC assay showed within- and between-run mean accuracy between 99.7% and 107% and coefficients of variation ≤6.7%. Matrix effects were nonsignificant, and samples were stable for 3 months preanalytically when stored at -80°C. TAC concentrations in the renal core biopsies were 62.6 and 43.7 pg/mg tissue. The methods for measurement of desmethyl-TAC and P-gp expression were suitable for semiquantification in homogenates from renal core biopsies.
These methods may be valuable for the elucidation of the pharmacokinetic mechanisms behind TAC-induced nephrotoxicity in renal transplant recipients.
Mycophenolic acid (MPA) mediates immunosuppressive effects by inhibiting inosine monophosphate dehydrogenase (IMPDH). Induction of IMPDH activity has been observed in whole blood and erythrocyte ...samples during immunosuppressive therapy. Information concerning the mechanisms for increased IMPDH activity is limited and the potential implications of induction have been debated.
Whole blood, CD4+ cell, and reticulocyte samples were collected from 30 renal transplant patients pre- and posttransplantation. The expressions of two IMPDH isoforms, type 1 and 2, were analyzed by real-time reverse-transcription polymerase chain reaction and quantified using a housekeeping gene index. The IMPDH activity was determined by ultraviolet high-performance liquid chromatography.
Transplantation and the initiation of immunosuppressive therapy was associated with increased IMPDH1 (50-88%, P<0.0005) and decreased IMPDH2 (42-56%, P<0.0005) expression. In CD4+ cells, however, IMPDH2 increased (15%, P=0.009). These changes are probably related to glucocorticoid effects. Two weeks posttransplant, MPA-treated patients displayed elevated IMPDH 1 and 2 in reticulocytes, suggesting enzyme induction in these cells during prolonged MPA therapy. Patients with acute rejection during follow-up demonstrated higher IMPDH2 expression in CD4+ cells pretransplant than nonrejecting patients (median expression 1.26 vs. 0.87 respectively, P=0.017).
Knowledge of changes in IMPDH 1 and 2 expression after transplantation and initiation of immunosuppression is important considering the action of MPA on IMPDH and the potential for pharmacodynamic monitoring of MPA by measuring IMPDH activity. The expression of IMPDH2 in CD4+ cells pretransplant may be an indicator of immune activation.
Abstract
Objective. The immunosuppressants cyclosporine, tacrolimus, sirolimus and everolimus are used in rejection prophylaxis after transplantation. Liquid chromatography tandem mass spectrometry ...(LC-MS/MS) has become a widely used methodology for monitoring of the drug levels to ensure therapeutic exposure. The main objective of the study was to evaluate the existence and potential influence of matrix effects on LC-MS/MS measurements of the immunosuppressants in clinical blood samples. Methods. The samples were prepared by protein precipitation and thereafter analysed by reversed-phase chromatography coupled to MS/MS via an electrospray interface. Assay performance including within- and between-series imprecision and deviations from external controls were examined. Elution of overall matrix components and glycerophosphocholines were investigated. The MS/MS signals were monitored in post-column infusion experiments, and post-precipitation addition of compounds provided a basis for quantification of the matrix effects. The influence of matrix effects on assay performance was investigated after dilution of quality controls with blood from multiple individuals. Results. Between-series coefficients of variation were ≤ 5.1, ≤ 6.6, ≤ 11.0 and ≤ 7.4 %, and the mean deviations from external controls were −10.3, −6.7, 15.6 and 4.3% for cyclosporine, tacrolimus, sirolimus and everolimus, respectively. The elution of matrix components including glycerophosphocholines overlapped to some extent with the target compounds, and the average ion suppression ranged from 8.5-21%. However, the drugs and internal standards were correspondingly influenced. Conclusion. The internal standards consistently corrected the between-individual variability of matrix effects. These findings consolidate the reliability of the assay.
Glucocorticoids are a group of steroid hormones with immunosuppressive and anti-inflammatory properties. In this article, we report the development and the validation of a liquid chromatography ...tandem mass spectrometry method for the simultaneous quantification of prednisolone, prednisone, cortisol, cortisone, methylprednisolone, and dexamethasone in human plasma. Furthermore, matrix effects were assessed qualitatively and quantitatively.
Plasma protein precipitation was performed with acetonitrile containing internal standards. Liquid-liquid extraction with dichloromethane and evaporation were used for cleanup and enrichment. The glucocorticoids were analyzed using reversed-phase chromatography and multiple reaction monitoring of positive ions.
The mean extraction recovery was in the range 66.5%-104.8%, whereas the lower limits of quantification ranged from 1.5 to 4.0 μg/L. The intraday and interday accuracies of all the analytes were within 89.4%-116.6%, and imprecision was <15.6%. Ion suppression ranged from 15.3% to 27.3%. However, the matrix effects did not compromise the assay performance, with mean deviations in calculated concentrations of -4.8% to 2.1% between methanol and matrix. Short-term stability was acceptable for 5 of the analytes, with deviations from baseline between -3.4% and 8.7% after 24 hours at 4°C, although methylprednisolone was stable for 6 hours with a degradation of 10.2%. Deviations from baseline in controls stored at -20°C for 6 months ranged from -22.3% to 6.3%. All analytes were stable after 3 repetitive freeze-thaw cycles, with a maximum degradation of 5.5%. In terms of postpreparative stability, the analytes were stable after 24 and 48 hours at 4°C, with maximum degradation of 6.1% and 9.4%, respectively.
A validated, sensitive, selective, and reproducible method for quantifying the concentrations of 6 glucocorticoids in human plasma by liquid chromatography tandem mass spectrometry is reported.
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