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The development of new and efficient adsorbents for dispersive solid-phase extraction method, particularly prior to chromatography analysis, is increasing. In particular, this method is recommended for use before biological sample analysis. In this work, a new composite was prepared from mesoporous carbon nitrides and carbon nano-onions and was utilized for the extraction of tacrolimus and everolimus from plasma samples prior to high-performance liquid chromatography-tandem mass spectrometry analysis. To achieve this aim, first, mesoporous carbon nitrides and carbon nano-onions were synthesized separately and mixed at optimized proportions. Subsequently, a suitable amount of the prepared composite (5 mg) was added to 2 mL of sample solution containing the analytes under vortexing. Next, the extracted analytes were eluted using acetonitrile. The approach was linear within the ranges of 1.0-500 and 0.51-500 ng mL -1 for tacrolimus and everolimus, respectively. Sensitive limits of detection (0.31 and 0.15 ng mL -1 for tacrolimus and everolimus, respectively), acceptable relative standard deviations (intra- and inter-day precisions of ≤5.6% and high extraction recoveries of 71.0% and 83.0% for tacrolimus and everolimus, respectively) were obtained. The results showed that the method can be successfully applied in the simultaneous extraction of the studied analytes from plasma.

The mTOR-inhibitor everolimus is a precision drug with antiepileptogenic properties approved for treatment of epilepsy in persons with tuberous sclerosis complex (TSC) in combination with other antiseizure medications (ASMs). However, the pharmacokinetic variability of everolimus is scarcely described, and the available information on pharmacokinetic interactions is scarce. The purpose of this study was to investigate pharmacokinetic variability of everolimus in patients with TSC, and the impact of age, sex and comedication. In this retrospective observational study we used anonymized data from medical records of patients with TSC using everolimus in Norway and Denmark, 2012 to 2020. Long-term therapeutic drug monitoring (TDM) identified inter-patient and intra-patient variability. The study included 59 patients, (36 females (61%)), median age 22 (range 3-59 years). Polytherapy was used in 50 patients (85%). The most frequently used ASMs were lamotrigine (n = 21), valproate (n = 17), and levetiracetam (n = 13). Blood concentrations of everolimus were measured in all patients. Pharmacokinetic variability of everolimus between patients was extensive, as demonstrated by a 24-fold variability from minimum-maximum concentration/dose (C/D)-ratios. The coefficient of variation (CV) for intra-patient (n = 59) and inter-patient variability (n = 47, ≥3 measurements) was 40% and 43%, respectively. The C/D-ratio of everolimus was 50% lower in 13 patients (22%) using enzyme-inducing ASMs compared to the 30 patients who did not (0.7 vs 1.4 ng/mL mg, P < .05). Age and sex were not significantly associated with changes in C/D-ratios of everolimus. Long-term TDM identified extensive variability in concentrations over time for everolimus both within and between patients, where comedication with enzyme-inducing ASMs was an important contributing factor. The findings suggest a need for TDM in patients with TSC treated with everolimus., Competing Interests: The other authors have no conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Everolimus is used for immunosuppression after renal transplantation. This study aimed to develop a population pharmacokinetic (PopPK) model of everolimus using therapeutic drug monitoring (TDM) data of patients under long-term multiple immunosuppressive therapy, including tacrolimus. To develop the model, 185 renal transplant recipients with 3358 everolimus blood concentrations during a median postoperative period of 35.3 months were included. The PopPK model is described as a one-compartment model with first-order absorption. The population mean of apparent clearance is 8.92 L/h (relative standard error = 3.6%), and this negatively correlated with the dose-normalized concentration (C/D) of tacrolimus and hematocrit value, and positively correlated with a daily dose of everolimus (i.e. TDM effect). The usefulness of dose adjustment using the final popPK model was assessed by a simulation study. The ratio of the first trough measurement within the therapeutic range of 3-8 ng/mL increased from 69.8% in the original dose to 87.9% in the individual dose calculated by the final PopPK model. The tacrolimus C/D ratio before initiating everolimus therapy and the hematocrit value were useful to estimate the initial dose of everolimus and can improve the safety and effectiveness of immunosuppressive therapy involving everolimus., (Copyright © 2024 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

A fast and reliable QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method for pre-processing combined with Ultra - high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) was established for the analysis of five mammalian rapamycin target protein (mTOR) inhibitors (vistusertib, AZD8055, pictilisib, everolimus, temsirolimus)in human plasma. Extraction was achieved by addition of acetonitrile to the sample followed by anhydrous magnesium sulfate and 30 mg C18 for salting out and purification, respectively. MTOR inhibitors were detected using selective response monitoring (SRM) under positive ion electrospray mode. Vistusertib, AZD8055 and pictilisib showed good linearity with a range of 1-80 ng/ml, Additionally, the concentration of everolimus and temsirolimus was 2.5-200 ng/ml and10-800 ng/ml, respectively. The linear correlation coefficient (R 2 ) of each analysis was ≥ 0.9950. The limit of detection (LOD) and Limit of Quantitation (LOQ) were 0.015-0.75 ng/ml and 1-10 ng/ml, respectively. This method showed a high accuracy with high recovery rate and excellent stability. This method is fast, accurate and reliable, suitable for quantitative detection of mTOR inhibitors in human plasma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The aim of this study was to develop and validate a UHPLC-MS/MS assay to quantify cyclosporin (CYC), tacrolimus (TAC), sirolimus (SIR) and everolimus (EVE) in human whole blood for therapeutic drug monitoring. Analytes were extracted from 50 μL human whole blood by protein precipitation. The separation of the drugs was performed on an Acquity UPLC BEH C18 column. Analytes were eluted with a mobile phase consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in deionised water and 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol at a flow rate of 300 μL/min in gradient elution. The method performance was evaluated by analysing patient blood samples and/or external quality control samples [proficiency testing (PT) scheme]. The method was linear from 23.75 to 1094.0, 1.3 to 42.4, 1.3 to 47.0 and 1.2-41.6 μg/mL for CYC, TAC, SIR and EVE, respectively. The within- and between-assay reproducibility results were ˂ 11%. Results from PT and patient sample quantification were comparable to those obtained previously by an in-house validated method using protein precipitation and liquid-liquid extraction. This method showed good analytical performance for quantifying CYC, TAC, SIR and EVE in whole blood over their respective calibration ranges., (© 2021 John Wiley & Sons, Ltd.)

Purpose: Therapeutic drug monitoring (TDM) is widely used in clinical practice to maximize drug efficacy and minimize toxicities. Currently, it is also practiced in the use of oral molecular targeted drugs. The objective of this study was to assess the clinical importance of measuring the systemic concentration of oral molecular targeted drugs used to treat renal cell carcinoma (RCC)., Methods: The systemic concentrations of the oral molecular targeted drugs sorafenib, sunitinib, axitinib, pazopanib, and everolimus used for RCC were useful for therapeutic interventions, and clinical outcomes were evaluated retrospectively., Results: The interventional use of systemic drug concentration was confirmed in 26 of 87, and their categories are presented. The systemic concentration of sunitinib was useful in dose reduction and/or discontinuation (n = 10), dose escalation (n = 3), and adherence monitoring (n = 2). Nine of the 10 patients whose dose was reduced showed reduced adverse event. Two patients who were intervened in adherence monitor showed improved adherence. For axitinib, dose reduction and/or discontinuation (n = 1) and dose escalation (n = 6) were confirmed. For pazopanib, dose reduction and/or discontinuation (n = 1) and drug interaction detection (n = 1) were confirmed, both of them were confirmed to have reduced adverse events. For everolimus, dose reduction and/or discontinuation (n = 1) and drug interaction detection (n = 1) were confirmed, a patient with reduced dose recovered from adverse events. Interventions for sorafenib were not identified., Conclusions: This study demonstrated that systemic concentrations of oral molecular targeted drugs for RCC were considered to be clinically useful for dose adjustment, monitoring of treatment adherence, and the detection of drug interactions. Moreover, this information could be successfully used to guide individualized therapy to maximize the antitumor effects of these drugs.

Background: Therapeutic drug monitoring of the immunosuppressants tacrolimus, sirolimus, everolimus, and cyclosporine A is effectively performed by analyzing whole-blood samples using liquid chromatography coupled with tandem mass spectrometry. Samples are usually prepared using simple protein precipitation (PPT) with methanol and zinc sulfate (ZnSO4). Significant sample dilution is necessary to obtain clean extracts but may increase the limit of quantification of the method. Salting out-assisted liquid-liquid extraction (SALLE) was explored as a novel sample preparation method for measuring these drugs in blood., Method: SALLE, which simply consists of LLE with a water-miscible solvent where phase separation is achieved by adding salt, was used to analyze treated blood samples., Results: SALLE allowed direct injection of a 5-µL extract from the upper solvent phase into a reversed phase LC column, which would not be feasible using standard LLE. Compared with PPT, SALLE provided better extraction efficiencies and more ion enhancement, resulting in limit of quantification of 0.4, 1.4, 0.06, and 0.4 ng/mL for tacrolimus, sirolimus, everolimus, and cyclosporine A, respectively. Full-method validation was performed, including a comparison of results with those of another laboratory. A ≤10% bias was observed for tacrolimus and cyclosporine A, whereas further investigation of that for sirolimus (-12%) and everolimus (-18%) revealed that it was caused by the different calibrators used., Conclusions: This is the first report of the use of SALLE for the measurement of tacrolimus, sirolimus, everolimus, and cyclosporine A in whole blood. The advantages of SALLE over PPT and conventional LLE would make it an attractive sample preparation method for clinical laboratories.

Genetic variations in CYP3A4, CYP3A5, and m-TOR could contribute to interpatient variability regarding m-TOR inhibitors pharmacokinetics or cellular effects. The purpose of this study was to evaluate the influence of selected candidate variations in these genes on everolimus pharmacokinetics, efficacy, and toxicity in cancer patients. Thirty-four patients receiving everolimus for breast (n = 22) or renal (n = 10) cancers, or neuroendocrine tumors of pancreatic origin (n = 2) were included in the study. Six variants in genes related to everolimus pharmacokinetics (CYP3A4*22 and CYP3A5*3) or pharmacodynamics (m-TOR rs2295079, rs2295080, rs2024627 and rs1057079) were genotyped. Associations with trough concentrations (C 0 ), dose reductions, or treatment interruptions due to toxicity and progression-free survival were investigated using generalized estimating equations and Cox models. CYP3A5 nonexpressers had significantly higher C 0 as compared with expressers (βGG vs AG = + 6.32 ± 2.22 ng/mL, p = 0.004). m-TOR rs2024627 was significantly associated with an increased risk of cancer progression studied alone or as part of an haplotype (T vs C: HR = 2.60, 95% CI [1.16-5.80], p = 0.020; CTCG vs other haplotypes HR = 2.29, 95% CI [1.06-4.95], p = 0.035, respectively). This study showed that CYP3A5 expression impacts everolimus pharmacokinetics in cancer patients and identified a genetic variation in m-TOR associated with the risk of cancer progression.

An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and 13 C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus (k = 2)., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

No data are available on rivaroxaban use in renal transplant recipients and on its surmised interaction with immunosuppressants. The aim was to investigate potential interactions between rivaroxaban and immunosuppressants in this setting. Renal transplant recipients with a stable renal function treated with rivaroxaban and tacrolimus with or without everolimus were investigated. All drugs and creatinine concentrations were determined daily for 2 weeks after the start of anticoagulation. Blood samples were drawn at 8.00 am and 3-4 h later for trough and peak concentrations, respectively. Bleeding and thrombotic events were recorded during a minimum follow-up of 6 months. In 8 renal transplant patients, rivaroxaban levels showed a predictable pharmacokinetic trend, both at C trough (30-61 μg/L) and at C peak (143-449 μg/L), with limited variability in the 25th-75th percentile range. Tacrolimus (C trough 3-13 μg/L; C peak 3-16 μg/L), everolimus (C trough 3-11 μg/L; C peak 5-17 μg/L) and creatinine concentrations were stable as well. Immunosuppressors variability before and after rivaroxaban were 30% and 30% for tacrolimus, 27% and 29% for everolimus, respectively, as well as 14% and 3% for creatinine. For rivaroxaban monitoring, the reference change value better performed in identifying significant variations of its concentration. No patient had bleeding or thrombotic events, worsening of renal graft function, and signs of immunosuppressants toxicity during a mean follow-up of 23 (9-28) months. In conclusion, rivaroxaban does not seem to interact with tacrolimus and everolimus in renal transplant recipients. Both anticoagulant and immunosuppressive effects seem warranted, without major bleeding complications and effect on the graft function., Competing Interests: Declaration of Competing Interest None of the authors reports a substantial direct or indirect commercial or financial incentive associated with publication of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

According to the standard ISO 15189 clinical routine laboratories shall estimate measurement uncertainty (MU) of patient results of their provided measurands. Up to now there was no accepted description on how to perform. Recently, the ISO technical standard ISO/TS 20914 was published giving a practical guide for uncertainty estimation. The immunosuppressive drugs Everolimus, Ciclosporin, Sirolimus and Tacrolimus have narrow therapeutic windows. Hence, their MU should be considered for deducing clinical decisions. Here, a pathway is presented in detail on how to estimate MU measuring immunosuppressants using a widespread CE certified assay via LC-MS/MS technology. Namely, the expanded measurement uncertainties are from 13% to 27% depending on analyte and concentration. The calculation based on n  > 2000 measurements each of four control levels within one year. Lower uncertainties were observed if the material was native pooled blood (13% to 17%, n  > 300 measurements, one year).

Measurement of immunosuppressive drug concentrations cyclosporine A (CyA), tacrolimus (TAC), sirolimus (SIR), and everolimus (EVE) in blood is an important application of therapeutic drug monitoring. These immunosuppressive agents are used in combined regimens and nowadays the liquid chromatography and tandem mass spectrometry is the best option for simultaneous analysis of these drugs in one short run. We developed an liquid chromatography and tandem mass spectrometry methodology in-house to measure the combination of immunosuppressants in a single blood sample from transplant patients in Brazil. We analyzed 235 combinations of 4 immunosuppressive drugs in patient blood to validate this study. The measuring ranges were 9 to 1000 ng/mL for CyA and 2 to 50 ng/mL for TAC, SIR, and EVE. Accuracy of the method was between 83.87% and 126.6% (coefficient of determination [r 2 ] > 0.995). Validation of variation was ≤15% for lower limit of quantification. In our analysis 20% of patients treated with EVE showed concentration range of 6 to 6.9 ng/mL, 28% of patients treated with SIR showed a concentration range of 4 to 4.9 ng/mL to TAC, 22% of patients showed concentration range of 5 to 5.9 ng/mL, and finally 50% of patients treated with CyA showed concentration range of 20 to 30 ng/mL. Our routine laboratory was able to implement this new methodology in-house to measure simultaneous CyA, TAC, SIR, and EVE in a single blood sample from transplant patients with a sensibility and rapid quantification analysis., (Copyright © 2020. Published by Elsevier Inc.)

Immunosuppressant medications help suppress the immune system response through inhibition of various checkpoints in the regulatory biochemical pathway. This is useful in prevention of organ rejection in transplantation or in the treatment of autoimmune diseases such as lupus or rheumatoid arthritis. Quantification of immunosuppressive drugs in blood is needed clinically for optimization of treatment and to avoid toxicity or unwanted side effects. Here, we describe a quantitative method to determine the concentration of cyclosprine A, tacrolimus, sirolimus, and everolimus in whole blood. This method has been used for many years clinically to support patient care. © 2020 by John Wiley & Sons, Inc., (© 2020 John Wiley & Sons, Inc.)

Background: Laboratory-developed tests (LDTs) are analytical tests developed and validated "in-house" for clinical diagnosis. Regulatory agencies, such as the United States Food and Drug Administration (FDA), encourage using regulatory-approved assays rather than LDTs. In the ongoing Zortracker everolimus study, samples were provided monthly to participating clinical laboratories that conduct therapeutic drug monitoring of everolimus. This allowed for the long-term (6-year) comparison of the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays, which are LDTs, to the FDA-approved everolimus Quantitative Microsphere System (QMS)., Methods: Each laboratory received the same 3 blinded samples. LC-MS/MS and QMS assays were compared using Passing Bablok regression analysis. Data were analyzed in 12-month periods to detect trends over time., Results: The slopes of the Passing Bablok regression curves remained unchanged in 2013 and 2014 (reference LC-MS/MS; test QMS: slope = 0.934 and 1.008). However, by 2016, the slope increased significantly to between 1.111 and 1.320, then dropped to 0.980 in 2017 and 0.912 in 2018, suggestive of changes in QMS bias compared with LC-MS/MS over longer periods. Outliers did not affect these results. The interlaboratory variability of LC-MS/MS and QMS remained unchanged from 2013 to 2015, with coefficients of variation of 15.3%, 18.4%, and 17.2% for LC-MS/MS, and 13.0%, 13.1%, and 15.3% for QMS, respectively, per year. At the end of the observation period, the average coefficients of variation in LC-MS/MS laboratories dropped to 14.3%, 12.6%, and 14.2%, whereas the variability in QMS laboratories was 19.5%, 13.3%, and 19.6% in 2016, 2017, and 2018, respectively., Conclusions: Initially, QMS everolimus concentrations in patient samples were comparable with those detected in LC-MS/MS laboratories. However, concentration bias of the QMS assay significantly changed within 6 years, emphasizing the need for long-term, independent performance tracking of therapeutic drug monitoring assays, including FDA-approved assays.

Objective: To evaluate the clinical and radiographic follow-up of renal angiomyolipoma (AML) in pediatric patients with tuberous sclerosis complex (TSC) on mTOR inhibitors., Methods: We performed retrospective chart review of children who were diagnosed with TSC between 2000 and 2019 and prescribed everolimus at age ≤18 years. Treatment assessment was performed in patients who were medically-compliant by serum drug trough levels and who had at least a baseline and one subsequent renal imaging study., Results: Nineteen patients were analyzed. Average age of everolimus initiation was 9 years, and indication was neurologic in 17 (90%). Fourteen patients (73.6%) had AML with average size of 1.9 (0.4-5) cm. Medication was discontinued due to side effects in 3 (16%) patients. Treatment assessment was analyzed for 15 patients with median medication exposure 5.1 (0.8-8.5) years. Among 13 with AML, the dominant lesion decreased in size in 9 (69%) and stayed stable in 4 (31%). Greatest absolute size decrease was seen for lesions ≥2 cm. No new AML lesions formed during treatment., Conclusion: Although not currently approved for this indication, everolimus appears to be well-tolerated with similar efficacy for pediatric AML as in adult AML. Use may be most warranted in children with AML ≥2 cm., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The mechanistic target of rapamycin inhibitors everolimus and sirolimus have activity against multiple manifestations of tuberous sclerosis complex and are approved to treat astrocytomas, angiomyolipomas, lymphangioleiomyomatosis, and epilepsy. Cannabidiol is a novel antiepileptic medication. There is lack of information regarding drug-drug interactions between mechanistic target of rapamycin inhibitors and cannabidiol in clinical practice., Methods: We reviewed patients with tuberous sclerosis complex who were treated with a mechanistic target of rapamycin inhibitor (everolimus, sirolimus) and cannabidiol. Clinical information, mechanistic target of rapamycin inhibitor and cannabidiol dosing, concomitant antiepileptic drugs, as well as laboratory and adverse events were reviewed before and after initiation of cannabidiol., Results: A total of 25 patients were treated with cannabidiol and a mechanistic target of rapamycin inhibitor (18 everolimus, seven sirolimus). All mechanistic target of rapamycin inhibitor levels were drawn as troughs. Levels were significantly higher in 76% patients after cannabidiol treatment (P = 0.0003). Median change from baseline was +9.8 ng/mL for everolimus and +5.1 ng/mL for sirolimus. Adverse events occurred in 40%, with diarrhea being the most frequent adverse event occurring in three patients. No severe adverse events occurred during the treatment period., Conclusions: Cannabidiol resulted in increased serum levels of everolimus and/or sirolimus. Some patients experienced doubling or tripling of their mechanistic target of rapamycin inhibitor trough following the addition of cannabidiol. In some cases, this resulted in clinical toxicity, as well as laboratory abnormalities. Awareness of this interaction can lead clinicians to evaluate serum levels and other safety laboratory studies more closely, and thereby avoid potentially significant adverse effects. In patients known to be prone to mechanistic target of rapamycin inhibitor toxicity, preemptive reduction in dose may be warranted upon initiation of cannabidiol., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background Monitoring of immunosuppressive drugs such as everolimus and sirolimus is important in allograft rejection prevention in transplant patients. Dried blood spots (DBS) sampling gives patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. Methods A total of 39 sirolimus and 44 everolimus paired fingerprick DBS and whole blood (WB) samples were obtained from 60 adult transplant patients for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. Two validation limits were pre-defined: limits of analytical acceptance were set at >67% of all paired samples within 20% of the mean of both samples and limits of clinical relevance were set in a multidisciplinary team at >80% of all paired samples within 15% of the mean of both samples. Results For both sirolimus and everolimus, Passing-Bablok regression showed no differences between WB and DBS with slopes of 0.86 (95% CI slope, 0.72-1.02) and 0.96 (95% CI 0.84-1.06), respectively. Only everolimus showed a significant constant bias of 4%. For both sirolimus and everolimus, limits of analytical acceptance were met (76.9% and 81.8%, respectively), but limits or clinical relevance were not met (77.3% and 61.5%, respectively). Conclusions Because pre-defined limits of clinical relevance were not met, this DBS sampling method for sirolimus and everolimus cannot replace WB sampling in our center at this time. However, if the clinical setting is compatible with less strict limits for clinical relevance, this DBS method is suitable for clinical application.

Background: Immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) are two major methods for therapeutic drug monitoring (TDM) of immunosuppressant drugs. Compared to the relatively limited analytical performance and cross reactivities of immunoassays, the LC-MS/MS method is considered as a gold standard; however, the lack of systematic evaluation and standardization needs to be addressed., Methods: A LC-MS/MS method for the determination of cyclosporine A, sirolimus, tacrolimus, and everolimus was developed. One-step protein precipitation was used to prepare blood samples. The newly developed method was systematically evaluated and validated according to the standard guidelines., Results: The quantitative method for four immunosuppressant drugs in human whole blood was validated according to the guidelines. The lower limits of the measuring interval (LLMI) for cyclosporine A, sirolimus, tacrolimus, and everolimus were 5, 0.5, 0.5, and 0.5 ng/mL, respectively. Linear correlation coefficients were all >0.999. Internal standard-normalized (IS-normalized) matrix correction factor was within the range 0.88-1.17. The average spiked recoveries of five replicates for the four immunosuppressant drugs were in the range 87.4-109.6%., Conclusion: An LC-MS/MS method combined with one-step protein precipitation was developed, providing short sample preparation and chromatographic run time, thus allowing easy clinical diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Everolimus (EVR) is often administered with cyclosporine A (CsA), according to an established protocol. Although the administration protocol of EVR with tacrolimus (TAC) has not been established, it has been clinically demonstrated that a higher dose of EVR is necessary when used in combination with TAC than with CsA. In this study, we aimed to determine the optimal dose of EVR administered with TAC to maintain a similar EVR level in the blood to that observed when EVR is administered with CsA. Between June 2009 and January 2016, 22 patients who underwent living donor kidney transplantation were enrolled in this study. Among them, 12 patients were administered steroids, basiliximab, CsA, and EVR (CsA + EVR group) and 10 were administered steroids, basiliximab, TAC, and EVR (TAC + EVR group). Blood samples were collected at different time points from patients in both CsA + EVR and TAC + EVR groups, after drug administration. The trough EVR level in both groups was maintained within 3-8 ng/mL during the perioperative period. The optimal EVR doses for both groups were estimated by using a population pharmacokinetic analysis. Overall, the optimal dose of EVR for the TAC + EVR group was 3.59-fold higher than that for the CsA + EVR group to maintain a similar trough level to that of the latter group. Thus, administration of a higher EVR dose is recommended when provided in combination with TAC than with CsA to prevent adverse events caused by under immunosuppression, that could lead to acute kidney rejection., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background Dried blood spots (DBSs) could allow patients to prepare their own samples at home and send them to the laboratory for therapeutic drug monitoring (TDM) of immunosuppressants. The purpose of this review is to provide an overview of the current knowledge about the impact of DBS-related preanalytical factors on TDM of tacrolimus, sirolimus and everolimus. Content Blood spot volume, blood spot inhomogeneity, stability of analytes in DBS and hematocrit (Hct) effects are considered important DBS-related preanalytical factors. In addition, the influence of drying time has recently been identified as a noteworthy preanalytical factor. Tacrolimus is not significantly influenced by these factors. Sirolimus and everolimus are more prone to heat degradation and exhibited variations in recovery which were dependent on Hct and drying time. Summary and outlook DBS-related preanalytical factors can have a significant impact on TDM for immunosuppressants. Tacrolimus is not significantly influenced by the studied preanalytical factors and is a viable candidate for DBS sampling. For sirolimus and everolimus more validation of preanalytical factors is needed. In particular, drying conditions need to be examined further, as current protocols may mask Hct-dependent effects on recovery. Further validation is also necessary for home-based self-sampling of immunosuppressants as the sampling quality is variable.

Purpose: Everolimus, a mammalian target of rapamycin inhibitor, may have a protective role on hepatocellular carcinoma (HCC) recurrence after liver transplantation (LT), but data regarding the impact of its trough serum levels on HCC recurrence are missing., Methods: Fifty-five patients (43 men, age 55 ± 8 years) who underwent LT for HCC were evaluated. Several demographic and clinical variables were recorded, including radiological and histological characteristics of HCC as well as dosages and trough levels of immunosuppressive regimens., Results: HCC recurrence occurred in 11 (20%) patients: 5 (25%) of 20 patients under calcineurin inhibitors and 6 (17%) of the 35 patients under everolimus (P = .48). The patients with HCC recurrence (n = 11, group 1), compared to those without recurrence (n = 44, group 2), had significantly more frequent HCC in the explant: outside Milan criteria (P = .001), microvascular invasion (P < .001), and higher number of nodules (P = .001). In multivariate analysis, microvascular invasion was the only independent factor significantly associated with HCC recurrence (OR: 2.3, 95% CI: 1.4-10.5, P = .03). Among the patients who received everolimus-based immunosuppression, the recipients with HCC recurrence, compared to those without HCC recurrence, had significantly lower mean trough levels of everolimus at 7-12 months post-LT (3.9 vs 5.9 ng/mL, P = .001), while the patients with mean trough levels of everolimus >6 ng/mL had decreased HCC recurrence rates (log rank: 2.3, P = .007)., Conclusions: We found for the first time mean concentrations of everolimus between 7-12 months post-LT as the only modifiable variable related with HCC recurrence in LT recipients. However, larger studies are needed for final conclusions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Many studies demonstrate the relationship between the high intrapatient variability of calcineurin inhibitor (CNI) levels and poor long-term renal graft outcome. Our objective is to analyze the intrapatient variability observed in the mammalian target of rapamycin inhibitors (mTOR-i) blood levels, to compare the variability of sirolimus (SRL) with that of everolimus (EVL) in kidney transplant patients converted to an mTOR-i, and to analyze whether the coefficient of variation (CV) was correlated with long-term graft survival., Methods: We analyzed 279 adult renal transplant patients converted to an mTOR-i. CV was calculated using at least 3 blood trough levels between 3 and 18 months postconversion., Results: The mean and median CV of the entire group was 25.54% and 23.7%, respectively. SRL and EVL mean CV was 23.8% and 27.1% (P = .03), respectively. The group of patients into the last tertile with CV> 28.52% presented a lower death-censored graft survival (75.26% vs. 93.01%, P < .0001) with a mean follow-up of 66.5 months., Conclusion: The CV of mTOR-i is correlated with long-term renal graft survival, so it should be considered a prognostic factor. SRL has a lower CV than EVL in renal transplant patients converted to mTOR-i in the stable posttransplant phase., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Everolimus is a mammalian target of rapamycin inhibitor approved for the treatment of various tumor types. Less invasive measurement of everolimus concentrations could facilitate pharmacokinetic studies and personalized dosing based on whole blood concentrations, known as therapeutic drug monitoring. Volumetric Absorptive Microsampling (VAMS) has been introduced as a patient friendly, less invasive sampling technique to obtain an accurate volume of whole blood regardless of hematocrit value. We describe the bioanalytical validation and clinical application of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify everolimus using VAMS. For the quantification, 13 C 2 D 4 -Everolimus was used as internal standard (IS). Everolimus and the IS were extracted with methanol from the VAMS device, which was evaporated after ultrasonification and shaking. The residue was reconstituted in 20 mM ammonium formate buffer and methanol (50%, v/v) of which 5 μL was injected into the LC-MS/MS system. Quantification was performed for the ammonium adduct of everolimus in positive electrospray ion mode. The VAMS method met all pre-defined validation criteria. Accuracy and precision were within 11.1% and ≤14.6%, respectively. Samples were shown to be stable on the VAMS device for at least 362 days at ambient temperatures. Considerable biases from -20 to 31% were observed over a 30-50% hematocrit range. Although the method fulfilled all validation criteria, the perceived advantage of VAMS over dried blood spot sampling could not be demonstrated. Despite the effect of hematocrit, using an empirically derived formula the whole blood everolimus concentration could be back calculated with reasonable accuracy in the clinical application study., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Objective: Intracellular exposure of everolimus may be a better marker of therapeutic effect than trough whole blood concentrations. We aimed to develop pharmacokinetic population models and Bayesian estimators based on a limited sampling strategy for estimation of dose interval exposures of everolimus in whole blood and peripheral blood mononuclear cells (PBMCs) in renal transplant recipients., Methods: Full whole blood and PBMC concentration-time profiles of everolimus were obtained from 12 stable renal transplant recipients on two different occasions, 4 weeks apart. The dataset was treated as 24 individual profiles and split into a development dataset (n = 20) and a validation dataset (n = 4). The pharmacokinetic model was developed using non-parametric modeling and its performances and those of the derived Bayesian estimator were evaluated in the validation set., Results: A structural two-compartment model with first-order elimination and two absorption phases described by a sum of two gamma distributions were developed. None of the tested covariates (age, sex, albumin, hematocrit, fat-free mass and genetic variants such as CYP3A5*1, ABCB1 haplotype, PPARA*42, PPARA*48, and POR*28) were retained in the final model. A limited sampling schedule of two whole blood samples at 0 and 1.5 h and one PBMC sample at 1.5 h post dose provided accurate estimates of the area under the plasma concentration-time curve (AUC) in comparison with the trapezoidal reference AUC (relative bias ± standard deviation = - 3.9 ± 10.6 and 4.1 ± 12.3% for whole blood and PBMC concentrations, respectively)., Conclusion: The developed model allows simultaneous and accurate prediction of everolimus exposure in whole blood and PBMCs, and supplies a base for a feasible exploration of the relationships between intracellular exposure and therapeutic effects in prospective trials.

What Is Known and Objective: For analysis of blood concentrations of everolimus, many hospital laboratories use either latex agglutination turbidimetric immunoassay (LTIA) or electrochemiluminescence immunoassay (ECLIA). However, no studies have compared both immunoassay methods under the same conditions. Accordingly, in this study, we compared everolimus blood concentrations obtained by LTIA and ECLIA in renal transplant patients., Methods: Blood samples (n = 230) from 60 renal transplant patients (19 female and 41 male) were evaluated using both immunoassays. Subsequently, we switched the assay for detection of everolimus blood concentrations from LTIA to ECLIA as a clinical application. Three quality control (QC) samples for LTIA were analysed using ECLIA, and 3 QC samples for ECLIA were analysed using LTIA., Results: The Deming regression of ECLIA versus LTIA generated the following parameters: slope, 1.0067 and intercept, 1.7489 ng/mL, in the analysis of 230 samples. Bland-Altman analysis showed an average positive bias of 1.73 ng/mL between ECLIA and LTIA. When the clinical apparatus was switched from LTIA to ECLIA, the average everolimus blood concentration assayed by LTIA before switching was 3.57 ng/mL, whereas that by ECLIA after switching in the same patients taking the same daily dose (mean: 1.43 mg/day) was 5.85 ng/mL. The QCs assayed using LTIA were lower by an average of 67.3% (range: 55.8%-79.5%) for ECLIA, and in the same 230 samples from patients, the everolimus blood concentrations assayed by LTIA were lower by an average of 67.4% (range: 37.1%-114.5%) of ECLIA., What Is New and Conclusion: Analysis of everolimus concentrations by immunoassays with high precision and accuracy is required to ensure long-term survival of transplant recipients. Although the concentrations of QCs and calibrators of everolimus in LTIA were previously corrected to 70% concentration because of cross-reactivity with everolimus metabolites, these adjustments may need to be reviewed., (© 2018 John Wiley & Sons Ltd.)

The mTOR signaling pathway has emerged as a possible therapeutic target for epilepsy. Clinical trials have shown that mTOR inhibitors such as everolimus reduce seizures in tuberous sclerosis complex patients with intractable epilepsy. Furthermore, accumulating preclinical data suggest that mTOR inhibitors may have anti-seizure or anti-epileptogenic actions in other types of epilepsy. However, the chronic use of rapalogs such as everolimus is limited by poor tolerability, particularly by immunosuppression, poor brain penetration and induction of feedback loops which might contribute to their limited therapeutic efficacy. Here we describe two novel, brain-permeable and well tolerated small molecule 1,3,5-triazine derivatives, the catalytic mTORC1/C2 inhibitor PQR620 and the dual pan-PI3K/mTOR inhibitor PQR530. These derivatives were compared with the mTORC1 inhibitors rapamycin and everolimus as well as the anti-seizure drugs phenobarbital and levetiracetam. The anti-seizure potential of these compounds was determined by evaluating the electroconvulsive seizure threshold in normal and epileptic mice. Rapamycin and everolimus only poorly penetrated into the brain (brain:plasma ratio 0.0057 for rapamycin and 0.016 for everolimus). In contrast, the novel compounds rapidly entered the brain, reaching brain:plasma ratios of ∼1.6. Furthermore, they significantly decreased phosphorylation of S6 ribosomal protein in the hippocampus of normal and epileptic mice, demonstrating effective mTOR inhibition. PQR620 and PQR530 significantly increased seizure threshold at tolerable doses. The effect of PQR620 was more marked in epileptic vs. nonepileptic mice, matching the efficacy of levetiracetam. Overall, the novel compounds described here have the potential to overcome the disadvantages of rapalogs for treatment of epilepsy and mTORopathies directly connected to mutations in the mTOR signaling cascade., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Few years ago, it was proposed that everolimus blood levels could be determined with the commercially available sirolimus chemiluminescence magnetic microparticle immunoassay (CMIA). More recently, a highly specific microsphere system (QMS) has been approved by FDA for therapeutic drug monitoring in humans. Aim of the present study was to compare the results of everolimus assay performed with everolimus QMS and with sirolimus CMIA. The two methods were compared with Passing-Bablok regression and Bland-Altman plot analysis. The Passing-Bablok regression analysis showed that although the results obtained with the two techniques were significantly correlated, CMIA-measured differed from QMS-measured everolimus concentrations by both a systematic and a proportional error. Specifically, at blood levels lower than 5 ng/mL CMIA were lower than QMS-measured everolimus concentrations. On the opposite, at everolimus blood concentrations higher than 10 ng/mL CMIA-estimated values became progressively higher than QMS-measured everolimus concentrations. The analysis of the Bland Altman plot showed a less than optimal agreement of the two tests (5.59% of the data point outside the ±1.96 SD interval). Moreover, the relationship between the difference between Evero QMS and Evero CMIA and their average was clearly concentration dependent with positive and negative values at concentration values lower and higher than 5 ng/mL respectively. In conclusion, our finding showed that the values of everolimus concentrations measured with sirolimus CMIA differ from those detected with the FDA-approved everolimus QMS further suggesting that sirolimus CMIA should not be used anymore for everolimus therapeutic drug monitoring.

Purpose: Everolimus treatment is seriously hampered by its toxicity profile. As a relationship between everolimus exposure and effectiveness and toxicity has been established, early and ongoing concentration measurement can be key to individualize the dose and optimize treatment outcomes. Dried blood spot (DBS) facilitates sampling at a patients' home and thereby eases dose individualization. The aim of this study is to determine the agreement and predictive performance of DBS compared to whole blood (WB) to measure everolimus concentrations in cancer patients., Methods: Paired DBS and WB samples were collected in 22 cancer patients treated with everolimus and analyzed using UPLC-MS/MS. Bland-Altman and Passing-Bablok analysis were used to determine method agreement. Limits of clinical relevance were set at a difference of ± 25%, as this would lead to a different dosing advice. Using DBS concentration and Passing-Bablok regression analysis, WB concentrations were predicted., Results: Samples of 20 patients were suitable for analysis. Bland-Altman analysis showed a mean ratio of everolimus WB to DBS concentrations of 0.90, with 95% of data points within limits of clinical relevance. Passing-Bablok regression of DBS compared to WB revealed no constant bias (intercept 0.02; 95% CI 0.93-1.35) and a small proportional bias (slope 0.89; 95% CI 0.76-0.99). Predicted concentrations showed low bias and imprecision and 90% of samples had an absolute percentage prediction error of < 20%., Conclusions: DBS is a valid method to determine everolimus concentrations in cancer patients. This can especially be of value for early recognition of over- or underexposure to enable dose adaptations.

While tacrolimus and everolimus have common metabolic pathways through CYP3A4/5, tacrolimus is metabolized solely by CYP3A4 in recipients with the CYP3A5*3 / *3 . The purpose of this study was to evaluate how the area under the blood concentration-time curves (AUC) of tacrolimus could be predicted based on CYP3A5 genotype and the AUC of everolimus in renal transplant patients taking both drugs. The dose-adjusted AUC (AUC/D) of tacrolimus and everolimus were calculated at one month and one year after transplantation. Significant correlations between the AUC/D of tacrolimus and everolimus were found for patients with the CYP3A5*1 allele or CYP3A5*3 / *3 at both one month and one year. At both stages, the determination coefficients were higher and the slopes of regression equations were larger for patients with CYP3A5*3 / *3 compared to the CYP3A5*1 allele. A good correlation between single doses of tacrolimus and everolimus was found for CYP3A5*3 / *3 patients at 1 year after transplantation ( r = 0.794, p < 0.001). The variability of the AUC 0-24 /D of tacrolimus for each CYP3A5 genotype could be predicted based on the AUC 0-12 /D of everolimus. Clinicians may be able to comprehensively carry out the dose adjustments of tacrolimus and everolimus based on relationship with AUCs of both drugs in each CYP3A5 genotype., Competing Interests: The authors declare no conflict of interest.

Appropriate exposure to posaconazole (PSZ) has been limited until the recent approval of the delayed-release oral tablet formulation. Our goal was to determine the exposure obtained by using the standard dose of 300 mg once a day in lung transplant (LT) patients, including patients with cystic fibrosis (CF). PSZ trough concentrations ( C 0 ) were determined using a liquid chromatography-tandem mass spectrometry assay. Indicative thresholds of interest were <0.7 mg/liter for prophylaxis and 1 to 3 mg/liter for cure. The tacrolimus (TRL) and everolimus (ERL) C 0 measured during PSZ exposure were also collected. The interaction with proton-pump inhibitors (PPI) was evaluated. We recorded the results for 21 CF patients with LT (CFLT patients), 11 non-CF patients with LT (NCFLT patients), and 27 nontransplant (NT) patients in pneumology departments. The weights of the NCFLT, CFLT, and NT patients were 59.2 ± 8.4, 48.8 ± 8.4, and 63.7 ± 16.6 kg, respectively ( P = 0.001* [asterisk means that statistical test is significant]), and the PSZ C 0 exposures for these patients were 1.9 ± 1.5, 1.1 ± 0.8, and 2.4 ± 1.8 mg/liter, respectively ( P < 0.00001*). More than 60% of the concentrations were in the therapeutic range. In CFLT patients, the administration of one 300-mg PSZ tablet quickly achieved an exposure similar to that achieved with the PSZ oral suspension formulation (OSF) administered 3 or 4 times a day for several months. The TRL C 0 /dose ratio ( C 0 / D ) was 7.4 ± 4.4 mg/liter with PSZ tablets, whereas it was 4.6 ± 0.8 mg/liter with the PSZ oral solution ( P = 0.034*). The ERL C 0 / D was similar with both formulations. PPI had no impact on the PSZ concentration (1.49 ± 1.07 mg/liter without PPI versus 1.33 ± 1.17 mg/liter with PPI; P = 0.4134*). Despite the high levels of exposure, PSZ remained well tolerated (one case of diarrhea and one case of fatigue were reported). PSZ tablet administration allows satisfactory exposure, even in CFLT patients, with a dosage lower than that of the PSZ OSF. This once-a-day formulation was not impacted by PPI, which are extensively used in CF patients., (Copyright © 2018 American Society for Microbiology.)

While the therapeutic drug monitoring (TDM) of everolimus has been routinely performed for over 10 years in solid organ transplantation medicine, in order to optimize the balance between effectiveness and toxicity, it is yet uncommon in the treatment of malignancies. The aim of this study was to develop and validate a bioanalytical method to quantify everolimus in dried blood spots (DBS) to facilitate TDM for the oncology outpatient setting. The hematocrit effect of everolimus was investigated. An 7.5mm disk from the central part of the DBS was punched, followed by the extraction of everolimus from the DBS by methanol/acetonitrile (80/20%) spiked with deuterium-labelled everolimus as internal standard. Subsequently, everolimus was separated and analyzed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The UPLC-MS/MS method was validated according to the European Medicine Agency (EMA) guideline. Everolimus concentrations could be quantified over the range of 3-75μg/L. The intra- and inter-assay precision and accuracy of the method were shown to be acceptable (coefficient of variation ≤10.7% and relative error ≤4.4%, respectively). The matrix effects appeared to be influenced by the hematocrit effect. The hematocrit effect was tested in a range of 0.20-0.50L/L, at which hematocrit accuracy and precision were satisfactory at values ≥0.25L/L. However, at 0.20L/L hematocrit in combination with high everolimus concentrations of 20 and 40μg/L, the precision was adequate (≤7.4%), but the accuracy was >15% of the nominal concentration. Everolimus was stable in DBS for at least 80days at 2-8°C. Given these results, the everolimus DBS method has been successfully developed and validated. Special attention is necessary for cancer patients with both a 0.20L/L hematocrit in combination with everolimus concentrations ≥20μg/L. A clinical validation for the use of everolimus DBS in cancer patients is currently being undertaken., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The precise monitoring of everolimus, an immunosuppressant drug, is vital for transplant recipients due to its narrow therapeutic range. This study evaluated the analytical performance of a new electrochemiluminescence immunoassay (ECLIA) for everolimus concentrations in whole blood., Methods: Accuracy, imprecision, and sensitivity studies for the Roche Elecsys everolimus ECLIA were performed at 5 European laboratories. The ECLIA was compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, as well as the Quantitative Microsphere System everolimus assay., Results: Everolimus ECLIA accuracies were within the range 100% ± 9%. Coefficients of variation (CVs) across the target range were ≤4.8% for repeatability and ≤8.4% for intermediate imprecision, whereas multisite reproducibility at lower (2.71 mcg/L) and higher everolimus concentrations (3.0-30.0 mcg/L) resulted in CVs of ≤13.7% and ≤12.4%, respectively. The CV at the assay's lower limit of quantification without considering bias was excellent, estimated as ≤9.3% at 0.5 mcg/L. The weighted Deming regression analysis, used for comparison of the results obtained by everolimus ECLIA and by LC-MS/MS methods, yielded a slope of 1.21 [95% confidence interval (CI): 1.15-1.26], intercept of 0.478 mcg/L (95% CI: 0.241-0.716), and a Pearson correlation coefficient (r) of 0.91. A single-site comparison between the ECLIA and the Quantitative Microsphere System assay revealed a slope of 1.05 (95% CI: 0.917-1.17), intercept of 1.03 mcg/L (95% CI: 0.351-1.70), and r of 0.91., Conclusions: Based on these results, the Roche Elecsys everolimus ECLIA can be considered suitable for routine therapeutic drug monitoring. A positive bias was observed with respect to LC-MS/MS methods, suggesting that it may be necessary to rebaseline individual patients when switching from LC-MS/MS to the ECLIA; however, this must also be considered for any change of method for everolimus measurement.

Background: This study investigates the adequacy of initial everolimus (EVR) dose, with and without calcineurin inhibitors (CNI), in kidney transplant recipients., Methods: This retrospective cohort analysis involved data from 305 kidney transplant recipients participating in 3 randomized trials receiving reduced dose cyclosporin A (CsA) combined with EVR 0.75 mg BID (CSA/EVR0.75, N = 32) or 1.5 mg BID (CSA/EVR1.5, N = 31), reduced dose tacrolimus (TAC) combined with EVR 1.5 mg BID (TAC0.05/EVR1.5, N = 83), standard dose TAC combined with EVR 1.5 mg BID (TAC0.1/EVR1.5, N = 93), and EVR 1.5 mg BID (EVR1.5, N = 66) with TAC introduction after day 5. The adequacy of the initial EVR dose, based on EVR whole blood trough between 3 and 8 ng/mL, was compared using first EVR blood concentrations obtained at day 3 after transplantation., Results: Recipient age, proportion of patients with diabetes mellitus, and proportion of grafts from living donors were different among the groups. Dose-corrected EVR concentrations were higher in patients receiving CsA than in those receiving TAC or no calcineurin inhibitors (6.7 ± 5.9 versus 5.4 ± 2.2 versus 2.4 ± 0.8 versus 2.5 ± 0.9 versus 2.2 ± 0.7, P = 0.000). No differences were observed comparing dose adjusted EVR concentrations combined with TAC or alone (P = 0.073). The proportion of patients with EVR concentration below <3 ng/mL was lower when EVR was combined with CsA (25 versus 3 versus 43 versus 33 versus 50%, P = 0.000). Later introduction of TAC did not influence EVR concentrations. There were no differences in mean CsA concentrations comparing patients receiving EVR 0.75 or 1.5 mg BID (240 ± 143 versus 213 ± 105 ng/mL). On the other hand, mean TAC concentrations were higher according to the initial TAC dose regimen (6.4 ± 3.9 versus 9.8 ± 5.9 ng/mL)., Conclusions: In de novo kidney transplant recipients, the choice of the initial dose of EVR should consider the type of calcineurin inhibitor to reach target EVR concentration within the first week in a higher proportion of patients, maximizing the efficacy/toxicity profile.

Coated Blade Spray (CBS) is a technology that efficiently integrates sample preparation and direct coupling to mass spectrometry (MS) on a single device. In this article, we present CBS-tandem mass spectrometry (CBS-MS/MS) as a novel tool for the rapid and simultaneous determination of four commonly used immunosuppressive drugs (ISDs) in whole blood: tacrolimus (TAC) and cyclosporine-A (CycA), which are calcineurin inhibitors; and sirolimus (SIR) and everolimus (EVR), which are both mTOR (mechanistic target of rapamycin) inhibitors. Given that CBS extracts via free concentration, analytes that are largely bound to plasma proteins or red blood cells provide considerably lower extraction recovery rates. Therefore, we defy the solventless philosophy of SPME-based techniques, like CBS, by performing the analyte-enrichment step via direct immersion in a solvent-modified matrix. The assay was linear within the evaluated range of concentrations (between 1 and 100 ng/mL for EVR/SIR/TAC and 10-1000 ng/mL for CycA), and the limits of quantification were determined to be 10 ng/mL for CycA and 1 ng/mL for EVR/SIR/TAC. Good accuracy (87-119%) and linearity (r 2  ≥ 0.99) were attained over the evaluated range for all ISDs. Interassay imprecision (CV) determined from incurred sample reanalysis was ≤10% for all ISDs. Our method was validated using Liquichek™ whole blood immunosuppressant quality control (QC) standards purchased from Bio-Rad. Concentrations determined by CBS-MS/MS were inside the range specified by Bio-Rad and within 15% of the expected mean value for all ISDs at all QC levels. Furthermore, the effect of different hematocrit levels (20, 45, and 70%) in the entire calibration range was carefully studied. No statistical differences (RSD ≤ 7%) in the calibration curve slopes of ISDs in blood were observed. CBS offers a simpler workflow than that of traditional methods; it eliminates the need for chromatographic separation and provides a clean extract that allows for long-term MS instrumental operation with minimal maintenance. Additionally, because CBS integrates all analytical steps into one device, it eliminates the risk of instrumental carry-over and can be used as a low-cost disposable device for sample preparation and analysis. Fully-automated sample preparation simplifies the method and allows for total analysis times as short as 3 min with turn-around times of less than 90 min., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study describes a fast high performance liquid chromatography-diode array detection analytical methodology for quantification of tacrolimus, everolimus and cyclosporine A in whole blood samples, with minimum sample preparation steps. A short isocratic chromatographic elution was coupled with second-order calibration using multivariate curve resolution to stablish a smart and green methodology. Due to presence of matrix effect, a sample-added calibration strategy was used for quantification purposes. The serious issues related to background drift, chromatographic shifts and co-elution of non-calibrated blood components, were resolved by a proper background correction and multivariate curve resolution-alternating least squares (MCR/ALS) methods The main features of this study were based on the fact that the acquired data matrices were handled intelligently and all features of the concerned target analytes were taken into account. Satisfactory resolution and quantification results in the presence of matrix interferences were achieved and the second-order advantage was fully exploited. The average recoveries in therapeutic concentration ranges were 102±10%, 99±11% and 104±12% for TAC, EVR and CsA, with average relative prediction errors of less than 7%. Considering the advantages of the present strategy, such as increased selectivity, sensitivity and sufficiency of lower limit of quantification through multivariate advantage, simplicity of sample treatment steps, a fast elution pattern and also a low-cost instrumentation compared with LC-MS/MS, the proposed method has the significant merits as an alternative for simultaneous therapeutic monitoring of immunosuppressants., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Therapeutic drug monitoring (TDM) of immunosuppressive drugs (ISDs) with a narrow therapeutic index is an increasingly popular tool for minimizing drug toxicity while maximizing the prevention of graft loss and organ rejection. This review focuses on trends regarding analytical methods for the TDM of ISDs since 2011. The five most commonly prescribed immunosuppressive medications are critically reviewed: cyclosporine A, tacrolimus, sirolimus (rapamycin), everolimus, and mycophenolic acid. This review introduces the general background of TDM and ISDs and presents the recent developments in using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassays for the TDM of ISDs. Finally, a future perspective for these analytical methods is briefly discussed., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background: Although the proportion of elderly patients among renal transplant recipients has increased, pharmacokinetic (PK) studies of immunosuppressants rarely include older patients., Methods: We studied 12-hour everolimus (EVL) PK in 16 elderly renal transplant recipients (all whites; 10 men; mean age, 64 ± 2 years (61-71 years), in 4 separate timepoints (at 7, 30, 60, and 150 days) after EVL introduction, corresponding to a mean postrenal transplantation day: PK1 (43 ± 4 days), PK2 (65 ± 7 days), PK3 (106 ± 17 days), and PK4 (206 ± 40 days). Patients received EVL (target trough level (Ctrough, 3-8 ng/mL), prednisone, and tacrolimus (TCL) (target Ctrough, 2-5 ng/mL)., Results: Mean TCL-Ctrough was 7.2 ± 3.8, 4.9 ± 2.2, 4.9 ± 2.2, and 4.5 ± 1.2 ng/mL at PK1, PK2, PK3, and PK4, respectively. There were no differences among timepoints for mean EVL daily dose (data shown as PK3) (3.5 ± 1.3 mg/d), Ctrough (4.7 ± 2.5 ng/mL), AUC0-12h (106 ± 51 ng/h per mL), Caverage (8.8 ± 4.2 ng/mL), Cmax (19.2 ± 9.7 ng/mL), apparent Half-life (11.7 ± 4.2 hours), estimated total body clearance (0.39 ± 0.27 L/h), or fluctuation (166 ± 65%). Also, none of those PK parameters differed statistically when adjusted for body weight. EVL-Ctrough showed a very high correlation (r = 0.849) with AUC0-12h., Conclusions: Our data indicate that elderly renal transplant recipients starting EVL 1 month after transplantation along with a steady-state TCL level, present stable EVL-PK parameters without significant changes in dose or exposure during the first 6 months after renal transplantation.

Introduction: Therapeutic drug monitoring (TDM) of everolimus is not performed in oncology and no trough level (C 0 ) target has been yet defined. The aim of this study was to determine everolimus C 0 target for toxicity and efficacy., Materials and Methods: Clinical, biological and radiologic data from 54 patients were collected. Toxicity event was defined by termination, temporary interruption and/or dose reduction of everolimus while efficacy was defined as progression-free survival. C 0 values were dichotomized by ROC curve analysis and the association between exposure and outcome was determined using Cox models for repeated events (toxicity) or Cox model censured at the first event (progression free survival)., Results: Among the 42 patients (77.8%) with breast cancer, 10 (18.5%) kidney cancer and 2 (3.7%) neuroendocrine cancer, adverse events were reported in 75.9% of the patients (everolimus termination in 25.9% patients). C 0 everolimus higher than 26.3ng/mL (Sen=0.38,Spe=0.88) were associated with a 4-fold increased risk of toxicity (HR=4.12, IC95%=[1.48-11.5], p=0.0067) whereas C 0 lower than 11.9ng/mL were associated with a 3-fold increased risk of progression (HR=3.2, IC95%=[1.33-7.81],p=0.001)., Discussion: Further studies are required to evaluate the everolimus C 0 threshold proposed for toxicity (26.3ng/mL) and for progression (11.9ng/mL) especially with a large number of patients and more homogeneous types of cancer. However, these results are in favour of TDM for everolimus in oncology., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the RADIANT-2 trial, addition of everolimus to octreotide long-acting repeatable (LAR) exhibited a clinically meaningful 5.1-month improvement in progression-free survival (PFS) in patients with advanced functional neuroendocrine tumors. In this study, we characterized the effects of everolimus co-administration on octreotide LAR pharmacokinetics and its relationship with efficacy and safety. At least one evaluable blood everolimus and plasma octreotide predose minimum concentration (C min ) was available for 182 patients and 294 patients, respectively. Concomitant everolimus administration increased octreotide C min with a geometric mean ratio (everolimus/placebo) of 1.47 (90% confidence interval [CI] = 1.32-1.64). Risk for progression was consistently reduced when everolimus C min was increased twofold, regardless of octreotide exposure (hazard ratio [HR] = 0.74; 95% CI = 0.46-1.18; HR = 0.54; 95% CI = 0.32-0.92 for 6 ng/mL and 4 ng/mL octreotide, respectively). Risk for pulmonary or metabolic events was associated with increased everolimus C min . Co-administration of everolimus plus octreotide LAR increased octreotide C min , which did not impact efficacy., (© 2016 American Society for Clinical Pharmacology and Therapeutics.)

Background: Therapeutic drug monitoring is recommended to guide therapy with the immunosuppressant everolimus (EVL) in solid organ transplantation to prevent rejections and to limit toxicity. For therapeutic drug monitoring, predose EVL concentrations are measured in whole blood mainly by liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, 2 immunoassays [Quantitative Microsphere System (QMS) EVL and Elecsys EVL] are commercially available. The aim of this study was to evaluate the comparability of EVL results determined with the 2 immunoassays and a validated LC-MS/MS test using samples from kidney, liver, and heart transplant (KT, LT, and HT, respectively) recipients., Methods: Analysis of predose samples from KT (n = 56), LT (n = 60), and HT (n = 59) recipients, obtained at variable time points after transplantation, was performed by LC-MS/MS and with the 2 immunoassays. The QMS EVL assay was applied on Dimension Xpand Plus and the Elecsys EVL assay on cobas e 411 analyzer. Results were compared by the Spearman's rank correlation coefficient, unbiased Passing and Bablok linear regression test, and Bland-Altman plot., Results: Results generated with both immunoassays correlated well with those of LC-MS/MS. An overestimation of EVL concentrations by the Elecsys EVL compared with LC-MS/MS was observed (mean bias: 34.2%). Using the QMS EVL, a small but significant negative deviation (mean bias: -8.0%) was found. Looking at KT, HT, and LT samples separately, the bias to LC-MS/MS seen with the Elecsys EVL was similar. With the QMS EVL, the best agreement was observed with the KT samples followed by LT and HT., Conclusions: Results generated by the 3 methods are not consistent regarding their diagnostic value. Both laboratories and manufacturers should take care to inform their costumers about the between-method differences to avoid misinterpretation of the results in clinical practice.

Aim: Hematocrit (Ht) effects remain a challenge in dried blood spot (DBS) sampling. The aim was to develop an immunosuppressant DBS assay on two LC-MS/MS systems covering a clinically relevant Ht range without Ht correction., Results: The method was partially validated for tacrolimus, sirolimus, everolimus, cyclosporin A and fully validated for mycophenolic acid on an Agilent and Thermo LC-MS/MS system. Bias caused by Ht effects were within 15% for all immunosuppressants between Ht levels of 0.23 and 0.48 l/l. Clinical validation of DBS versus whole blood samples for tacrolimus and cyclosporin A showed no differences between the two matrices., Conclusion: A multiple immunosuppressant DBS method without Ht correction, has been validated, including a clinical validation for tacrolimus and cyclosporin A, making this procedure suitable for home sampling.

Tacrolimus (TAC, FK-506) and everolimus (EVE, RAD001) are immunosuppressors used to treat pediatric patients undergoing liver transplantation. Their hematic TDM by liquid chromatography became standard practice. However, it does not always reflect concentrations at their active site. Our aim was to develop and validate a new method for the simultaneous TAC and EVE quantification into target cells: peripheral blood mononuclear cells (PBMCs). Peripheral blood mononuclear cells were collected using cell preparation tubes; cells number and mean cell volume were evaluated by an automatic cell counter. TAC and EVE were quantified using UHPLC-MS/MS coupled with an automated online solid-phase extraction platform. Chromatographic run was performed on an Acquity UPLC® BEH C18 1.7 μm (2.1 × 50 mm) column at 45 °C, for 6 min at 0.5 ml/min. Mobile phases were water and methanol, both with 2 mm ammonium acetate and 1 ml/l formic acid). XBridge® C8 10 μm (1 × 10 mm) SPE cartridges were used, and the internal standard was ascomycin. Following Food and Drug Administration guidelines, method validation resulted in high sensitivity and specificity. Calibration curves were linear (r 2  = 0.998) and intra-day and inter-day imprecision and inaccuracy were <15%. A reproducible matrix effect was observed, with a good recovery for all compounds. Drug amounts in 15 'real' PBMCs samples from five pediatric patients in co-treatment resulted within the calibration range (0.039-5 ng). Concentrations from each patient were standardized using their evaluated mean cell volume: intra-PBMCs concentration was meanly 19.23 and 218.61 times higher than the hematic one for TAC and EVE, respectively. This method might be useful in clinical routine, giving reliable data on drugs concentration at the active site. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Context: Several assays of monitoring immune cell function have been developed to enhance therapeutic drug monitoring., Objective: An in vitro-validated whole-blood assay of phosphorylated ribosomal protein S6 (pS6RP) was evaluated for confounders to monitor the mTOR-inhibitor everolimus (ERL)., Materials and Methods: Whole blood samples from 87 heart transplant recipients were analyzed for pS6RP-expression in CD3-positive T-cells by phospho-flow analysis., Results: ERL blood concentration, laboratory parameters, co-medications, demographic and clinical data were reviewed., Conclusion: Evaluating the pS6RP-assay revealed that pS6RP is influenced by cyclosporine A (CsA) blood concentration, duration of ERL treatment, co-medication with thiazide diuretics and different metabolic parameters.

Objective: To evaluate circadian changes in everolimus (EVL) pharmacokinetics and to identify the time point of blood sampling with the strongest correlation with the area under the blood concentration-time curve (AUC) of EVL in heart transplant patients., Methods: Heart transplant patients receiving the same dose of EVL twice a day were reviewed. In 28 patients enrolled, whole blood samples were collected before (C0), and 1, 2, 4, 6, 8, and 12 hours after each administration of EVL. Blood concentrations of EVL were compared between active (9:00 AM to 9:00 PM) and resting periods (9:00 PM to 9:00 AM)., Results: AUC0-4h, peak concentration (Cmax), Cmax/minimum concentration, and peak-trough fluctuation in the resting period were significantly lower than those in the active period (p = 0.008, 0.017, 0.022, and 0.011, respectively). Half-life and mean residence time were significantly longer in the resting period than in the active period (p = 0.002 and 0.002, respectively). AUC0-12h in the active period was similar (p = 0.154) and correlated with that in the resting period (r 2  = 0.93). Two-point blood samplings, C0 and C2, correlated more strongly with AUC0-12h for EVL, compared with C0 alone (0.92 vs. 0.79, respectively, for r 2 in the active period)., Conclusions: EVL pharmacokinetics showed circadian changes, suggesting delayed absorption and decreased metabolic activity at rest. However, the circadian changes did not affect AUC0-12h. A 2-time-point model that included C0 and C2 was more accurate for predicting the AUC0-12h of EVL than C0 alone in heart transplant patients.
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Background: Everolimus has been used for the treatment of unresectable or metastatic renal cell carcinoma (RCC). Here, we measured blood concentrations of everolimus to obtain the population pharmacokinetic parameters and to examine the relationship between blood concentration and clinical outcomes., Methods: Twenty-two Japanese patients were enrolled. Blood samples were collected before and 2, 4, 8, and 24 hours after drug administration on days 1 and 8 of everolimus therapy (5 or 10 mg) from inpatients; occasional samples were collected from outpatients. Blood concentrations of everolimus were measured by high-performance liquid chromatography with tandem mass spectrometry. Population pharmacokinetic analysis was conducted using the NONMEM software., Results: Everolimus pharmacokinetics was best described by a 2-compartment model with population mean estimates of apparent oral clearance of 10.0 L/h and an interindividual variability of 42.4%. There was no relationship between overall best responses and the predicted trough concentrations at day 8. The predicted trough concentration in patients who terminated everolimus treatment owing to adverse drug reactions (ADRs) was significantly higher than in patients who stopped the treatment owing to disease progression or other reasons (27.6 ± 3.1 versus 15.7 ± 2.3 ng/mL; mean ± SEM). Patients who terminated the treatment owing to ADRs had significantly shorter time-to-treatment failure than other patients (112 versus 187 days, median)., Conclusions: This study reports the first population pharmacokinetic parameters of everolimus in patients with RCC. Individual dose adjustment based on everolimus blood concentrations helps to avoid early drug cessation due to ADRs.

Background: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) utilizing triple-quadrupole instruments has been widely used for quantification of endogenous compounds, drugs or metabolites in clinical laboratories. In contrast, high-resolution mass spectrometry (HRMS) is typically used for compound identification due to its limited dynamic range. Recently HRMS instruments with enhanced linear dynamic range have become available. The aim of this study was to evaluate HRMS for fast quantitative applications in a clinical laboratory., Methods: A high throughput UPLC-TOF-MS assay for simultaneous quantification of cyclosporin A, tacrolimus, sirolimus and everolimus was developed. All immunosuppressants were analyzed as sodium adducts in TOF-only mode using an Agilent 6540 Q-TOF system. Extracted ion chromatograms of analytes and internal standards were created from full-scan data. The assay was evaluated and compared to an established LC-MS/MS assay according to CLSI recommendations., Results: The novel HRMS assay has a total run time of 3 min. The assay is linear in a clinical relevant concentration range for all four immunosupressants. Method correlations vs. established LC-MS/MS assay were between R2=0.99 and R2=0.97. Total coefficients of variation (CVT) ranges were 4.5%-6.4% (tacrolimus), 7.4%-8.0% (sirolimus), 8.0%-8.8% (everolimus) and 6.1%-7.4% (cyclosporine A) for three relevant concentration levels each., Conclusions: High resolution TOF-MS and LC-MS/MS show equivalent quantitative performance for monitoring of cyclosporin A, tacrolimus, sirolimus and everolimus. HRMS has the potential to replace conventional LC-MS/MS in clinical laboratories because it simplifies assay development (no optimization of fragmentations and product ions necessary) and its full-scan data can provide additional information.

Measurement of whole blood sirolimus and everolimus is required in order to optimize patient treatment following solid organ transplant. Assay by LC-MS/MS is increasingly preferred; however efficient use of the instrument and short turnaround times are crucial. Use of a 1.6 µm solid-core packing HPLC column (Cortecs) gave significant increases in efficiency, sensitivity and throughput compared with an existing method, following simple protein precipitation of small-volume (20 μL) whole blood samples. Sirolimus, everolimus and the stable isotopic internal standard ((13) C2 D4 - everolimus) eluted at around 0.8 min, and total analytical run time was 2.2 min, saving almost 4 min per sample compared with an existing method. Within-assay imprecision (CV) was 3.3-8.5%, and between-assay imprecision was 2.2-10.8%. Retrospective assay of external quality assurance samples and comparison of patient samples assayed in parallel showed only small differences (between +6.8 and -1.9%) in results using the Cortecs column when compared with the existing method. No significant interferences or ion suppression were observed. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)