Generic Medicinal Products in Immunosuppressive Therapy-Should It be a Challenge for Therapeutic Drug Monitoring?

ABSTRACT: Immunosuppressants have a narrow therapeutic index (NTIDs). Indisputably cyclosporine, tacrolimus, everolimus, and sirolimus have NTIDs, and only in the case of mycophenolic acid, a scientific discussion has not been yet concluded. Their specificities highlight the implications for generics introduced into the drug market, more precisely, with bioequivalence testing. In the European Union, the European Medicines Agency (EMA) released the "Guideline on the Investigation of Bioequivalence." The bioequivalence (BE) of the generic (tested, T) versus original (reference, R) product should be confirmed by obtaining a 90% confidence interval (CI) for the T:R ratio of each of the 2 decisive pharmacokinetic parameters, namely, the area under the curve (AUC) between 90.00% and 111.11%. A similar approach (90.00%-112.00%) for AUC was adopted by the Canadian Agency for Drugs and Technologies in Health (CADTH) for NTIDs; however, the US Food and Drug Administration is still based on classic acceptance criteria: 90% CI between 80.00% and 125.00% but with special requirements of BE testing. A discussion about long-expected global consensus was performed in this study based on the literature concerning BE testing in the case of NTIDs. The narrow acceptance criteria reduce the potential mean difference in bioavailability between generic and original products by a few percent. To identify this problem, special attention has been paid to switching drugs (generic-generic, original-generic) and therapeutic drug monitoring after conversion (TDM). There is no global consensus on the acceptance criteria for the BE of generic drugs; therefore, consensus and harmonization are strictly necessary. This study presents a review of the generic drug market and its classification by manufacturers, drug agencies, and dates of marketing authorization. Guidelines for TDM optimization (during switching/conversion) have been proposed. Physicians and clinical pharmacists should pay special attention to switching immunosuppressive drugs between original versus generic formulations, and generic versus generic formulations. Patients and their families should be educated on the risks associated with uncontrolled conversion.

Therapeutic Drug Monitoring and Dosage Adjustments of Immunosuppressive Drugs When Combined With Nirmatrelvir/Ritonavir in Patients With COVID-19.

ABSTRACT: Nirmatrelvir/ritonavir (Paxlovid) consists of a peptidomimetic inhibitor (nirmatrelvir) of the SARS-CoV-2 main protease and a pharmacokinetic enhancer (ritonavir). It is approved for the treatment of mild-to-moderate COVID-19. This combination of nirmatrelvir and ritonavir can mediate significant and complex drug-drug interactions (DDIs), primarily due to the ritonavir component. Indeed, ritonavir inhibits the metabolism of nirmatrelvir through cytochrome P450 3A (CYP3A) leading to higher plasma concentrations and a longer half-life of nirmatrelvir. Coadministration of nirmatrelvir/ritonavir with immunosuppressive drugs (ISDs) is particularly challenging given the major involvement of CYP3A in the metabolism of most of these drugs and their narrow therapeutic ranges. Exposure of ISDs will be drastically increased through the potent ritonavir-mediated inhibition of CYP3A, resulting in an increased risk of adverse drug reactions. Although a decrease in the dosage of ISDs can prevent toxicity, an inappropriate dosage regimen may also result in insufficient exposure and a risk of rejection. Here, we provide some general recommendations for therapeutic drug monitoring of ISDs and dosing recommendations when coadministered with nirmatrelvir/ritonavir. Particularly, tacrolimus should be discontinued, or patients should be given a microdose on day 1, whereas cyclosporine dosage should be reduced to 20% of the initial dosage during the antiviral treatment. Dosages of mammalian target of rapamycin inhibitors (m-TORis) should also be adjusted while dosages of mycophenolic acid and corticosteroids are expected to be less impacted.

Identification of Novel CB2 Ligands through Virtual Screening and In Vitro Evaluation.

Cannabinoid receptor type 2 (CB2) is a very promising therapeutic target for a variety of potential indications. However, despite the existence of multiple high affinity CB2 ligands, none have yet been approved as a drug. Therefore, it would be beneficial to explore new chemotypes of CB2 ligands. The recent elucidation of CB2 tertiary structure allows for rational hit identification with structure-based (SB) methods. In this study, we established a virtual screening workflow based on SB techniques augmented with ligand-based ones, including molecular docking, MM-GBSA binding energy calculations, pharmacophore screening, and QSAR. We screened nearly 7 million drug-like, commercially available compounds. We selected 16 molecules for in vitro evaluation and identified two novel, selective CB2 antagonists with Ki values of 65 and 210 nM. Both compounds are structurally diverse from CB2 ligands known to date. The established virtual screening protocol may prove useful for hit identification for CB2 and similar molecular targets. The two novel CB2 ligands provide a desired starting point for future optimization and development of potential drugs.

Limited sampling strategy to predict free mycophenolic acid area under the concentration-time curve in paediatric patients with nephrotic syndrome.

In paediatric patients, there is no data on the recommended area under the concentration-time curve from 0 to 12 h (AUC0-12 ) for free mycophenolic acid (fMPA), which is the active form of the drug, responsible for the pharmacological effect. We decided to establish the limited sampling strategy (LSS) for fMPA for its use in MPA therapeutic monitoring in children with nephrotic syndrome treated with mycophenolate mofetil (MMF). This study included 23 children (aged 11 ± 4 years) from whom eight blood samples were collected within 12 h after MMF administration. The fMPA was determined using the high-performance liquid chromatography with fluorescence detection method. LSSs were estimated with the use of R software and bootstrap procedure. The best model was chosen based on a number of profiles with AUC predicted within ± 20% of AUC0-12 (good guess), r2 , mean prediction error (%MPE) of ±10% and mean absolute error (%MAE) of less than 25%. The fMPA AUC0-12 was 0.1669 ± 0.0697 µg h/mL and the free fraction was within 0.16%-0.81%. In total, there were 92 equations developed of which five fulfilled the acceptance criteria for %MPE, %MAE, good guess >80% and r2 > 0.900. These equations consisted of three time points: model 1 (C1 , C2 , C6 ), model 2 (C1 , C3 , C6 ), model 3 (C1 , C4 , C6 ), model 5 (C0 , C1 , C2 ), and model 6 (C1 , C2 , C9 ). Although blood sampling up to 9 h after MMF dosing is impractical, it is crucial to include C6 or C9 in LSS to assess fMPA AUCpred correctly. The most practical fMPA LSS, which fulfilled the acceptance criteria in the estimation group, was fMPA AUCpred  = 0.040 + 2.220 × C0 + 1.130 × C1 + 1.742 × C2 . Further studies should define the recommended fMPA AUC0-12 value in children with nephrotic syndrome.

Volumetric Absorptive Microsampling in Therapeutic Drug Monitoring of Immunosuppressive Drugs-From Sampling and Analytical Issues to Clinical Application.

Miniaturisation and simplification are novel approaches in clinical bioanalysis, especially in therapeutic drug monitoring (TDM). These contemporary trends are related to the sampling, pre-treatment, and analysis of biological fluids. Currently, dried blood spot (DBS), one of the most popular microsampling techniques, is feasible and inexpensive. However, obtaining reliable results with sample homogeneity and volume variability is difficult. Volumetric Absorptive Microsampling (VAMS) has recently enabled the accurate and precise collection of a fixed blood volume. It reduced the hematocrit effect, improved volumetric accuracy, and generated results correlating with the dose and drug exposure from wet blood. This review focuses on VAMS-Mitra™ devices, which have become increasingly important since 2014, mainly for TDM and toxicology studies. First, the current literature has been reviewed based on immunosuppressants and their determination in samples obtained using Mitra™. Second, the critical points, weaknesses, and strengths have been characterized in contrast to classic venipuncture and other microsampling methods. Finally, we indicate the points of attention according to the perspective of Mitra™ as well as its usefulness in clinical practice. VAMS is currently state-of-the-art in microsampling and seems to be a good instrument for improving adherence to immunosuppressive therapy, especially in the pediatric population.

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

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

A Guide to Targeting the Endocannabinoid System in Drug Design.

The endocannabinoid system (ECS) is one of the most crucial systems in the human organism, exhibiting multi-purpose regulatory character. It is engaged in a vast array of physiological processes, including nociception, mood regulation, cognitive functions, neurogenesis and neuroprotection, appetite, lipid metabolism, as well as cell growth and proliferation. Thus, ECS proteins, including cannabinoid receptors and their endogenous ligands' synthesizing and degrading enzymes, are promising therapeutic targets. Their modulation has been employed in or extensively studied as a treatment of multiple diseases. However, due to a complex nature of ECS and its crosstalk with other biological systems, the development of novel drugs turned out to be a challenging task. In this review, we summarize potential therapeutic applications for ECS-targeting drugs, especially focusing on promising synthetic compounds and preclinical studies. We put emphasis on modulation of specific proteins of ECS in different pathophysiological areas. In addition, we stress possible difficulties and risks and highlight proposed solutions. By presenting this review, we point out information pivotal in the spotlight of ECS-targeting drug design, as well as provide an overview of the current state of knowledge on ECS-related pharmacodynamics and show possible directions for needed research.

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

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

Pharmacokinetics of free and total mycophenolic acid in adult lupus nephritis patients-implications for therapeutic drug monitoring.

PURPOSE: To evaluate the relationship between total and free MPA pharmacokinetic (PK) parameters and renal outcome markers, and to verify whether conducting therapeutic drug monitoring (TDM) in lupus nephritis (LN) patients would be of value in routine clinical practice. METHODS: Eighty-four samples were collected from sixteen LN patients. Total and free MPA concentrations were measured at predose, 0.5 and 2 h after mycophenolate mofetil (MMF) intake. Area under the concentration time curve from 0 to 2 h (AUC0-2) and free fraction were calculated. RESULTS: High between-patient variability was observed (CV% of 53.5% for dose-normalized total MPA AUC0-2). A significant but weak correlation between dose-normalized total C0 and AUC0-2 was noted (r = 0.5699). Dose-normalized total C0 above 2.76 µg/mL·g may indicate patients with eGFR < 81 mL/min with sensitivity of 83.3% and specificity of 75.0%. Hypoalbuminemic LN patients demonstrated significantly elevated MPA free fraction when compared with patients with serum albumin concentration ≥ 3.5 g/dL (1.49 ± 0.64% vs 1.08 ± 0.75%). CONCLUSION: This study examined relationship between free and total pharmacokinetic MPA parameters as well as the effect of hypoalbuminemia on MPA plasma protein binding in adult LN patients. The study results suggest that TDM of MPA in LN seems to be a more reasonable approach than the fixed-dose protocol. Moreover, predose total MPA concentration may be a possible estimation of MPA exposure, while monitoring free rather than total MPA may be more beneficial in hypoalbuminemic patients.

Limited sampling strategy to predict mycophenolic acid area under the curve in pediatric patients with nephrotic syndrome: a retrospective cohort study.

PURPOSE: Limited sampling strategy (LSS) is a precise and relatively convenient therapeutic drug monitoring method. We evaluated LSSs for mycophenolic acid (MPA) in children with nephrotic syndrome treated with mycophenolic mofetil (MMF) and validated the LSSs using two different approaches. METHODS: We measured MPA plasma concentrations in 31 children using HPLC-UV method and received 37 MPA pharmacokinetic profiles (0-12 h). For six children, MPA profiles were estimated twice after two MMF doses. LSSs were developed using multilinear regression with STATISTICA and R software and validated using validation group and bootstrap method, respectively. RESULTS: The best three time point equations included C1, C3, C6 (good guess 83%, bias - 2.78%; 95% confidence interval (CI) - 9.85-0.46); C1, C2, C6 (good guess 72%, bias 0.72%; 95% CI - 5.33-7.69); and C1, C2, C4 (good guess 72%, bias 2.05%; 95% CI - 4.92-13.01) for STATISTICA software. For R software, the best equations consisted of C1, C3, C6 (good guess 92%, bias - 2.69%; 95% CI - 27.18-33.75); C0, C1, C3 (good guess 84%, bias - 2.11%; 95% CI - 24.19-22.29); and C0, C1, C2 (good guess 84%, bias - 0.48%; 95% CI - 30.77-54.07). During validation, better results were obtained for R evaluations, i.e., bootstrap method. CONCLUSIONS: The most useful equations included C0, C1, C3 and C0, C1, C2 time points; however, the most precise included C1, C3, C6 time points because of MPA enterohepatic recirculation. Better results were obtained for bootstrap validation due to greater number of patients. Validated LSS should be used only in the population for which it was developed. As there is growing evidence that underexposure of MPA is associated with insufficient treatment response, we recommend the introduction of therapeutic drug monitoring for MPA in children with nephrotic syndrome.

Free mycophenolic acid determination in human plasma ultrafiltrate by a validated liquid chromatography-tandem mass spectrometry method.

The aim of this study was to develop and validate fully the liquid chromatography-tandem mass spectrometry method for free mycophenolic acid (MPA) concentration measurements in plasma ultrafiltrate that will be reliable and simple in preparation with deuterated MPA (MPA-d3) chosen as an internal standard. The chromatographic separation was made with Zorbax Eclipse XDB-C18 column (4.6 × 150 mm) using a gradient of two solutions as a mobile phase: (A) water and (B) methanol, each containing 0.1% formic acid and 2.5 mm ammonium acetate. Satisfactory repeatability of retention times was achieved with average values of 7.54 ± 0.20 min and 7.50 ± 0.19 min for MPA and MPA-d3, respectively. The method was selective, with no carry-over or matrix effect observed. The analytical range was proven for MPA ultrafiltrate concentrations of 1-500 ng/mL. The accuracy and precision fell within the acceptance criteria for intraday (accuracy: 100.63-110.46%, imprecision: 6.23-7.76%), as well as interday assay (accuracy: 98.81-110.63%; imprecision: 5.36-10.22%). The method was used for free MPA determination in plasma samples from patients treated with mycophenolate mofetil. To the best of our knowledge this is the first liquid chromatography-tandem mass spectrometry method for free MPA monitoring using MPA-d3 that allows to measure plasma ultrafiltrate concentrations as low as 1 ng/mL.

DEVELOPMENT AND VALIDATION OF BROMATOMETRIC, DIAZOTIZATION AND VIS-SPECTROPHOTOMETRIC METHODS FOR THE DETERMINATION OF MESALAZINE IN PHARMACEUTICAL FORMULATION.

Three new methods were developed for the quantitative determination of mesalazine in the form of the pure substance or in the form of suppositories and tablets - accordingly: bromatometric, diazotization and visible light spectrophotometry method. Optimizing the time and the temperature of the bromination reaction (50°C, 50 min) 4-amino-2,3,5,6-tetrabromophenol was obtained. The results obtained were reproducible, accurate and precise. Developed methods were compared to the pharmacopoeial approach - alkalimetry in an aqueous medium. The validation parameters of all methods were comparable. Developed methods for quantification of mesalazine are a viable alternative to other more expensive approaches.

SIMULTANEOUS ASSAY OF ARIPIPRAZOLE AND ITS ACTIVE METABOLITE IN SERUM BY HPLC.

A rapid, convenient, precise HPLC method was developed for the simultaneous determination of aripiprazole and its active metabolite dehydroaripiprazole in blood serum. The separation was carried out by RP HPLC on Symmetry C18 column (150 x 4.6 mm; 5 µm). The mobile phase was composed of acetonitrile: water (30 : 70, v/v), pH 3.0 adjusted with o-phosphoric acid. The detection was monitored at 220 nm.

Barcelona Consensus on Biomarker-Based Immunosuppressive Drugs Management in Solid Organ Transplantation.

With current treatment regimens, a relatively high proportion of transplant recipients experience underimmunosuppression or overimmunosuppression. Recently, several promising biomarkers have been identified for determining patient alloreactivity, which help in assessing the risk of rejection and personal response to the drug; others correlate with graft dysfunction and clinical outcome, offering a realistic opportunity for personalized immunosuppression. This consensus document aims to help tailor immunosuppression to the needs of the individual patient. It examines current knowledge on biomarkers associated with patient risk stratification and immunosuppression requirements that have been generally accepted as promising. It is based on a comprehensive review of the literature and the expert opinion of the Biomarker Working Group of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology. The quality of evidence was systematically weighted, and the strength of recommendations was rated according to the GRADE system. Three types of biomarkers are discussed: (1) those associated with the risk of rejection (alloreactivity/tolerance), (2) those reflecting individual response to immunosuppressants, and (3) those associated with graft dysfunction. Analytical aspects of biomarker measurement and novel pharmacokinetic-pharmacodynamic models accessible to the transplant community are also addressed. Conventional pharmacokinetic biomarkers may be used in combination with those discussed in this article to achieve better outcomes and improve long-term graft survival. Our group of experts has made recommendations for the most appropriate analysis of a proposed panel of preliminary biomarkers, most of which are currently under clinical evaluation in ongoing multicentre clinical trials. A section of Next Steps was also included, in which the Expert Committee is committed to sharing this knowledge with the Transplant Community in the form of triennial updates.

Validated HPLC-UV method for determination of naproxen in human plasma with proven selectivity against ibuprofen and paracetamol.

Estimating the influence of interfering compounds present in the biological matrix on the determination of an analyte is one of the most important tasks during bioanalytical method development and validation. Interferences from endogenous components and, if necessary, from major metabolites as well as possible co-administered medications should be evaluated during a selectivity test. This paper describes a simple, rapid and cost-effective HPLC-UV method for the determination of naproxen in human plasma in the presence of two other analgesics, ibuprofen and paracetamol. Sample preparation is based on a simple liquid-liquid extraction procedure with a short, 5 s mixing time. Fenoprofen, which is characterized by a similar structure and properties to naproxen, was first used as the internal standard. The calibration curve is linear in the concentration range of 0.5-80.0 µg/mL, which is suitable for pharmacokinetic studies following a single 220 mg oral dose of naproxen sodium. The method was fully validated according to international guidelines and was successfully applied in a bioequivalence study in humans. Copyright © 2015 John Wiley & Sons, Ltd.

Therapeutic Drug Monitoring of Mycophenolic Acid in Lupus Nephritis: A Review of Current Literature.

In recent years, mycophenolate mofetil (MMF)-based immunosuppressive regimen was recommended in induction and in maintenance therapy in lupus nephritis (LN), one of the most severe and common manifestations of systemic lupus erythematosus. However, no recommendations were made so far regarding monitoring of mycophenolic acid (MPA) plasma concentrations. Therapeutic drug monitoring (TDM) constitutes a practical tool to ensure optimal posology. In renal transplantation, it was proved that acute allograft rejection incidences decreased when the recommended MPA target exposure has been maintained (30-60 mg·h·L). The results obtained in the field of transplant medicine indicate the potential benefit of carrying out TDM in LN. To date, the correlation of MPA exposure and clinical outcomes in the population of LN patients was the objective of just a few studies. The aim of this review was therefore to present TDM studies in LN patients on MMF therapy and to compare their results. Based on the conclusions drawn from TDM studies in LN, it can be suggested that the area under the concentration-time curve threshold values of 30-45 mg·h·L can potentially be associated with favorable treatment outcome. Moreover, the majority of the analyzed studies indicate relatively good correlation between trough concentration and the area under the concentration-time curve in patients treated with MMF that constitutes an important implication for TDM approach in routine setting. The threshold of 3 mg/L can potentially be recommended as a target trough value.

A Comparison of the Immunochemical Methods, PETINIA and EMIT, With That of HPLC-UV for the Routine Monitoring of Mycophenolic Acid in Heart Transplant Patients.

BACKGROUND: The aim of this study was to evaluate particle enhanced turbidimetric inhibition immunoassay (PETINIA) recently developed for mycophenolic acid (MPA) determination in plasma and to compare it with a reference high-performance liquid chromatography (HPLC) method, using samples from heart transplant recipients. The results are presented in the context of PETINIA being compared with enzyme multiplied immunoassay technique (EMIT). METHODS: PETINIA evaluation was performed using 194 routine trough plasma samples at steady state. EMIT was evaluated using 677 samples from 61 steady-state 12-hour profiles obtained from 35 heart transplant patients. Evaluation was undertaken on a Dimension EXL 200 analyzer (PETINIA) and on a Viva-E analyzer (EMIT). RESULTS: The mean MPA concentration measured by PETINIA was significantly higher than that measured by high-performance liquid chromatography combined with UV detector (2.36 ± 1.30 mcg/mL versus 1.82 ± 1.23 mcg/mL, respectively, P < 0.0001). Bland-Altman analysis revealed a mean bias of 0.54 mcg/mL [95% confidence interval (CI), 0.49-0.59] comprising 33.48% (95% CI, 30.34-36.61). Passing-Bablok regression was: y = 1.100x + 0.38 (95% CI for slope: 1.044-1.154 and for intercept: 0.30-0.47). Regardless of a significant observed correlation (r = 0.9230, P < 0.0001), the statistical analyses showed a significant difference between PETINIA and the reference chromatographic method. The mean MPA concentration measured by EMIT was significantly higher than that measured by HPLC (7.48 ± 8.34 mcg/mL versus 5.57 ± 6.61 mcg/mL, respectively, P < 0.0001) with a mean bias of 1.91 mcg/mL (95% CI, 1.75-2.07) comprising 35.91% (95% CI, 34.37-37.45). The significant difference between EMIT and HPLC was confirmed by Passing-Bablok regression: y = 1.300x + 0.24 (95% CI for slope: 1.279-1.324 and for intercept: 0.18-0.29). The analysis of the determinations, grouped by sampling time, revealed positive bias between EMIT and HPLC ranging from 24.54% to 42.77% and inversely proportional to MPA concentrations with r = 0.9122 (P < 0.001). CONCLUSIONS: The new immunochemical PETINIA method was associated with significantly higher MPA concentrations in routine therapeutic drug monitoring samples from heart transplant patients. The magnitude of the MPA overestimation was similar to that observed by use of the EMIT method.

A novel approach to therapeutic drug monitoring of Ciclosporin in pediatric renal transplant recipients using volumetric absorptive microsampling (VAMS) - Teaching old dog new tricks.

BACKGROUND AND AIMS: Ciclosporin (CSA) is an immunosuppressive agent that requires therapeutic drug monitoring (TDM). High partitioning in erythrocytes indicates that whole blood (WB) is a suitable matrix for CSA determination. Alternative sampling strategies, such as volumetric absorptive microsampling (VAMS), are novel possibilities for blood collection during TDM for various analytes, including immunosuppressants. This technique is attractive for vulnerable pediatric patients, including home-based self-sampling, remote therapy, and adherence control. MATERIALS AND METHODS: This study aimed to develop and validate a new method for CSA determination based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of WB and VAMS samples. Additionally, these methods were applied for CSA determination in clinical samples from pediatric transplant recipients. A strong point of this study is the assessment of an external proficiency testing scheme. RESULTS: Both methods were successfully validated within the 1 - 2000 ng/mL calibration range, with LOD 0.5 and 1 ng/mL for WB and VAMS methods, respectively. All the validation parameters fulfilled the international acceptance criteria for bioanalytical methods. Cross-validation confirmed the interchangeability of the LC-MS/MS method developed in this study. CONCLUSION: This study developed and validated novel methods for CSA determination in whole blood and VAMS using LC-MS/MS. Clinical validation and proficiency testing confirmed their utility in routine clinical practice.

Development and validation of limited sampling strategies for the estimation of mycophenolic acid area under the curve in adult kidney and liver transplant recipients receiving concomitant enteric-coated mycophenolate sodium and tacrolimus.

BACKGROUND: Mycophenolic acid (MPA) is widely used in solid organ transplantation. MPA absorption from enteric-coated mycophenolate sodium (EC-MPS) is delayed, which results in a delayed enterohepatic recirculation and subsequently higher and more variable MPA 12-hour trough concentration and tmax values. Therefore, MPA trough level monitoring cannot be used to monitor MPA exposure in patients who are given EC-MPS. The aim of the study was to develop and validate a limited sampling strategy (LSS) for accurate prediction of the 12-hour area under the concentration-time curve (AUC0-12h) for MPA in patients who receive concomitant EC-MPS and Tacrolimus (Prograf or Advagraf) within 196 months posttransplantation. According to our knowledge, the LSS for MPA AUC estimation using high-performance liquid chromatography to determine MPA concentrations in plasma samples of kidney and liver transplant patients receiving EC-MPS and Tacrolimus (Advagraf) has not been previously evaluated. METHODS: Seventy-four renal and liver transplant patients receiving EC-MPS and concomitant tacrolimus (either Prograf or Advagraf) provided a total of 74 pharmacokinetic profiles. MPA concentrations were measured using a validated high-performance liquid chromatography method for 9 plasma samples collected at predose and at 0.5, 1, 2, 3, 4, 6, 9, and 12 hours after the morning dose of EC-MPS after an overnight fast. LSS were developed and validated by stepwise multiple regression analysis with the use of a 2-group method (test, n = 37; and validation, n = 37). RESULTS: The 3 and 4 time point equations using C1h, C3h, C9h and C1h, C2h, C3h, C6h, respectively, were found to be superior to all other models tested. When these LSS models were tested in the validation group, the results were acceptable [for 3 time points equation: r = 0.824, percentage of prediction error: 6.32 ± 25.75, 95% confidence interval (CI): -40.71 to 79.76; percentage of absolute prediction error: 27.45 ± 29.89, 95% CI: 0.04-199.92, predictive performance, 71% of estimated AUCs comprised within 85%-115% of the measured full MPA AUC, natural logarithmic residuals (ln) mean ± SD: -0.03 ± 0.24; for 4 time points equation: r = 0.898, percentage of prediction error: 3.32 ± 18.26, 95% CI: -49.35 to 51.06; percentage of absolute prediction error: 14.05 ± 11.89, 95% CI 0.13-49.86, percentage of predictive performance, 83% of estimated AUCs comprised within 85%-115% of the measured full MPA AUC, natural logarithmic residuals (ln) mean ± SD: -0.01 ± 0.19]. CONCLUSIONS: LSS equations using concentrations at 1, 3, and 9 hours or 1, 2, 3, and 6 hours time points provided the most reliable and accurate estimations of the MPA AUC in stable renal and liver transplant recipients treated with EC-MPS and tacrolimus. Further studies on independent groups of patients are required to confirm clinical utility of the presented LSS models.