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.

Therapeutic drug monitoring of mycophenolic acid (MPA) using volumetric absorptive microsampling (VAMS) in pediatric renal transplant recipients: ultra-high-performance liquid chromatography-tandem mass spectrometry analytical method development, cross-validation, and clinical application.

BACKGROUND: Mycophenolic acid (MPA) is widely used in posttransplant pharmacotherapy for pediatric patients after renal transplantation. Volumetric absorptive microsampling (VAMS) is a recent approach for sample collection, particularly during therapeutic drug monitoring (TDM). The recommended matrix for MPA determination is plasma (PL), and conversion between capillary-blood VAMS samples and PL concentrations is required for the appropriate interpretation of the results. METHODS: This study aimed to validate and develop a UHPLC-MS/MS method for MPA quantification in whole blood (WB), PL, and VAMS samples, with cross and clinical validation based on regression calculations. Methods were validated in the 0.10-15 µg/mL range for trough MPA concentration measurement according to the European Medicines Agency (EMA) guidelines. Fifty pediatric patients treated with MPA after renal transplantation were included in this study. PL and WB samples were obtained via venipuncture, whereas VAMS samples were collected after the fingerstick. The conversion from VAMSMPA to PLMPA concentration was performed using formulas based on hematocrit values and a regression model. RESULTS: LC-MS/MS methods were successfully developed and validated according to EMA guidelines. The cross-correlation between the methods was evaluated using Passing-Bablok regression, Bland-Altman bias plots, and predictive performance calculations. Clinical validation of the developed method was successfully performed, and the formula based on regression was successfully validated for VAMSMPA to PLMPA concentration and confirmed on an independent group of samples. CONCLUSIONS: This study is the first development of a triple matrix-based LC-MS/MS method for MPA determination in the pediatric population after renal transplantation. For the first time, the developed methods were cross-validated with routinely used HPLC-DAD protocol.

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.

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.

Therapeutic Drug Monitoring of Tacrolimus Based on Volumetric Absorptive Microsampling Technique (VAMS) in Renal Transplant Pediatric Recipients-LC-MS/MS Method Development, Hematocrit Effect Evaluation, and Clinical Application.

Tacrolimus (TAC) is post-transplant pharmacotherapy's most widely used immunosuppressant. In routine clinical practice, frequent uncomfortable venipuncture is necessary for whole-blood (WB) collection to check trough TAC levels. Volumetric absorptive microsampling (VAMS) is an alternative strategy to WB collection. In this study, we aimed to validate and develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for TAC quantification in WB and VAMS samples. After extraction with water and protein precipitation, the samples were directly analyzed using LC-MS/MS. Whole-blood and VAMS capillary-blood samples were collected from 50 patients treated with TAC during the follow-up visits. The cross-correlation between the developed methods was evaluated using Passing-Bablok regression and a Bland-Altman bias plot. The matrix effect (ME) and carry-over were insignificant for both scenarios. There was a high correlation between the processes and no significant clinical deviation. LC-MS/MS methods were successfully developed and validated in the 0.5-60 ng/mL calibration range. This study demonstrated and confirmed the utility of VAMS-based TAC monitoring in the pediatric population. This is the first study to directly develop and validate the VAMS LC-MS/MS method for evaluating the hematocrit effect in the pediatric population. The statistical correlation between immunochemical and VAMS-based methods was satisfactory.

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.

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.

Development and Validation of the New Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Unbound Tacrolimus in the Plasma Ultrafiltrate of Transplant Recipients.

(1) Background: Only unbound tacrolimus particles are considered to be active and capable of crossing cellular membranes. Thus, the free-drug concentration might be better associated with clinical effects than the total drug concentration used for dosage adjustment. We propose a new, fully validated online liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for unbound tacrolimus concentration measurement. (2) Methods: The determination of the unbound tacrolimus concentration in plasma ultrafiltrate was performed with the Nexera LC system with LCMS-8050 triple quadrupole MS using ascomycin as an internal standard. Chromatographic separation was made using a HypurityC18 analytical column. MS/MS with electrospray ionization and positive-ion multiple-reaction monitoring was used. The unbound tacrolimus level was determined in 36 patients after solid organ transplantation (n = 140). (3) Results: A lower limit of quantification 0.1 pg/mL was achieved, and the assay was linear between 0.1 and 20 pg/mL (R2 = 0.991). No carry-over was detected. The within-run and between-run accuracies ranged between 97.8-109.7% and 98.3-107.1%, while the greatest imprecision was 10.6% and 10.7%, respectively. Free tacrolimus in patients' plasma ultrafiltrate varied between 0.06 and 18.25 pg/mL (median: 0.98 pg/mL). (4) Conclusions: The proposed method can be easily implemented. The significance of the unbound tacrolimus concentration needs to be investigated. This may facilitate the individualization and optimization of immunosuppressive treatment.

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.

Kidney and Liver Tissue Tacrolimus Concentrations in Adult Transplant Recipients-The Influence of the Whole Blood and Tissue Concentrations on Efficiency of Treatment during Immunosuppressive Therapy.

Tacrolimus (TAC) has a narrow therapeutic index and highly variable pharmacokinetic characteristics. Close monitoring of the TAC concentrations is required in order to avoid the risk of acute rejection or adverse drug reaction. The results in some studies indicate that inter-tissue TAC concentrations can be a better predictor with regards to acute rejection episode than TAC concentration in whole blood. Therefore, the aim of the study was to assess the correlation between dosage, blood, hepatic and kidney tissue concentration of TAC measured by a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) and clinical outcomes in a larger cohort of 100 liver and renal adult transplant recipients. Dried biopsies were weighed, mechanically homogenized and then the samples were treated with a mixture of zinc sulfate-acetonitrile to perform protein precipitation. After centrifugation, the extraction with tert-butyl methyl ether was performed. The analytical range was proven for TAC tissue concentrations of 10-400 pg/mg. The accuracy and precision fell within the acceptance criteria for intraday as well as interday assay. There was no correlation between dosage, blood (C0) and tissue TAC concentrations. TAC concentrations determined in liver and kidney biopsies ranged from 8.5 pg/mg up to 160.0 pg/mg and from 7.1 pg/mg up to 215.7 pg/mg, respectively. To the best of our knowledge, this is the first LC-MS/MS method for kidney and liver tissue TAC monitoring using Tac13C,D2 as the internal standard, which permits measuring tissue TAC concentrations as low as 10 pg/mg.

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.

Energetics of interactions in the solid state of 2-hydroxy-8-X-quinoline derivatives (X = Cl, Br, I, S-Ph): comparison of Hirshfeld atom, X-ray wavefunction and multipole refinements.

In this work, two methods of high-resolution X-ray data refinement: multipole refinement (MM) and Hirshfeld atom refinement (HAR) - together with X-ray wavefunction refinement (XWR) - are applied to investigate the refinement of positions and anisotropic thermal motion of hydrogen atoms, experiment-based reconstruction of electron density, refinement of anharmonic thermal vibrations, as well as the effects of excluding the weakest reflections in the refinement. The study is based on X-ray data sets of varying quality collected for the crystals of four quinoline derivatives with Cl, Br, I atoms and the -S-Ph group as substituents. Energetic investigations are performed, comprising the calculation of the energy of intermolecular interactions, cohesive and geometrical relaxation energy. The results obtained for experimentally derived structures are verified against the values calculated for structures optimized using dispersion-corrected periodic density functional theory. For the high-quality data sets (the Cl and -S-Ph compounds), both MM and XWR could be successfully used to refine the atomic displacement parameters and the positions of hydrogen atoms; however, the bond lengths obtained with XWR were more precise and closer to the theoretical values. In the application to the more challenging data sets (the Br and I compounds), only XWR enabled free refinement of hydrogen atom geometrical parameters, nevertheless, the results clearly showed poor data quality. For both refinement methods, the energy values (intermolecular interactions, cohesive and relaxation) calculated for the experimental structures were in similar agreement with the values associated with the optimized structures - the most significant divergences were observed when experimental geometries were biased by poor data quality. XWR was found to be more robust in avoiding incorrect distortions of the reconstructed electron density as a result of data quality issues. Based on the problem of anharmonic thermal motion refinement, this study reveals that for the most correct interpretation of the obtained results, it is necessary to use the complete data set, including the weak reflections in order to draw conclusions.

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.

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.

Isotope-labeled versus analog internal standard in LC-MS/MS method for tacrolimus determination in human whole blood samples - A compensation of matrix effects.

The aim of this work was to develop and to validate LC-MS/MS method for tacrolimus (TAC) determination in whole blood samples using two different types of internal standards (IS): an isotope-labeled TAC13C,D2 and a structural analog ascomycin (ASC). Matrix effects (ME) were evaluated to determine their influence on validation parameters. The LC-MS/MS analyses were performed using a 4000 QTRAP® mass spectrometer (AB Sciex) coupled to a HPLC 1260 Infinity system (Agilent Technologies). The [M + NH4]+ adducts were monitored with mass transitions of: 821.5 → 768.4 m/z for TAC, 809.5 → 756.4 m/z for ASC, and 824.6 → 771.5 m/z for TAC13C,D2. Blood samples were treated with 0.1 mol/L zinc sulfate - acetonitrile (50:50, v/v) then extracted with tert-butyl methyl ether. ME evaluations were performed by preextraction addition (n = 6), postextraction addition (n = 8), and repeated measures (n = 8) of reference solutions, separately for both ISs. ME, absolute recovery (AR) and process efficiency (PE) were calculated. Low (1.5 ng/mL) and high (16 ng/mL) TAC concentrations were tested. The method was successfully calibrated in a range of: 0.5-20 ng/mL. Both ISs provided satisfactory imprecision (<3.09% and <3.63% for TAC13C,D2 and ASC, respectively) and accuracy (99.55-100.63% and 97.35-101.71% for TAC13C,D2 and ASC, respectively). Similar ARs were found for all three compounds yielding: 74.89-76.36% for TAC, 78.37% for TAC13C,D2 and 75.66% for ASC. Significant ME were observed yielding on the average: -16.04% and -29.07% for TAC, -16.64% for TAC13C,D2 and -28.41% for ASC. Consequently, PE was 64.11% and 53.12% for TAC, 65.35% for TAC13C,D2 and 54.18% for ASC. ME for TAC were perfectly compensated (TAC/IS ratio) in each sample resulting in mean percent value of: 0.89% and -0.97% for TAC13C,D2 and ASC, respectively. An IS ascomycin presented a performance equivalent to TAC13C,D2 in LC-MS/MS method developed for TAC monitoring.

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.

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.