Circular Permutated PQQ-Glucose Dehydrogenase as an Ultrasensitive Electrochemical Biosensor.

Protein biosensors play an increasingly important role as reporters for research and clinical applications. Here we present an approach for the construction of fully integrated but modular electrochemical biosensors based on the principal component of glucose monitors PQQ-glucose dehydrogenase (PQQ-GDH). We designed allosterically regulated circular permutated variants of PQQ-GDH that show large (>10-fold) changes in enzymatic activity following intramolecular scaffolding of the newly generated N- and C termini by ligand binding domain/ligand complexes. The developed biosensors demonstrated sub-nanomolar affinities for small molecules and proteins in colorimetric and electrochemical assays. For instance, the concentration of Cyclosporine A could be measured in 1 µL of undiluted blood with the same accuracy as the leading diagnostic technique that uses 50 times more sample. We further used this biosensor to construct highly porous gold bioelectrodes capable of robustly detecting concentrations of Cyclosporine A as low as 20 pM and retained functionality in samples containing at least 60 % human serum.

Application of Design of Experiments® Approach-Driven Artificial Intelligence and Machine Learning for Systematic Optimization of Reverse Phase High Performance Liquid Chromatography Method to Analyze Simultaneously Two Drugs (Cyclosporin A and Etodolac) in Solution, Human Plasma, Nanocapsules, and Emulsions.

The objectives of current investigation are (1) to find out wavelength of maximum absorbance (λmax) for combined cyclosporin A and etodolac solution followed by selection of mobile phase suitable for the RP-HPLC method, (2) to define analytical target profile and critical analytical attributes (CAAs) for the analytical quality by design, (3) to screen critical method parameters with the help of full factorial design followed by optimization with face-centered central composite design (CCD) approach-driven artificial neural network (ANN)-linked with the Levenberg-Marquardt (LM) algorithm for finding the RP-HPLC conditions, (4) to perform validation of analytical procedures (trueness, linearity, precision, robustness, specificity and sensitivity) using combined drug solution, and (5) to determine drug entrapment efficiency value in dual drug-loaded nanocapsules/emulsions, percentage recovery value in human plasma spiked with two drugs and solution state stability analysis at different stress conditions for substantiating the double-stage systematically optimized RP-HPLC method conditions. Through isobestic point and scouting step, 205 nm and ACN:H2O mixture (74:26) were selected respectively as the λmax and mobile phase. The ANN topology (3:10:4) indicating the input, hidden and output layers were generated by taking the 20 trials produced from the face-centered CCD model. The ANN-linked LM model produced minimal differences between predicted and observed values of output parameters (or CAAs), low mean squared error and higher correlation coefficient values in comparison to the respective values produced by face-centered CCD model. The optimized RP-HPLC method could be applied to analyze two drugs concurrently in different formulations, human plasma and solution state stability checking.

Development of a Population Pharmacokinetic Model for Cyclosporine from Therapeutic Drug Monitoring Data.

AIM: To develop a population pharmacokinetic model for Uruguayan patients under treatment with cyclosporine (CsA) that can be applied to TDM. Patients and Methods. A total of 53 patients under treatment with CsA were included. 37 patients with at least one pharmacokinetic profile described with four samples were considered for model building, while the remaining 16 were considered for the assessments of predictive performances. Pharmacokinetic parameter estimation was performed using a nonlinear mixed effect modelling implemented in the Monolix® software (version 2019R1, Lixoft, France); meanwhile, simulations were performed in R v.3.6.0 with the mlxR package. RESULTS: A two-compartment model with a first-order disposition model including lag time was used as a structural model. The final model was internally validated using prediction corrected visual predictive check (pcVPC) and other graphical diagnostics. A total of 621 CsA steady-state concentrations were analyzed for model development. Population estimates for the absorption constant (ka) and lag time were 0.523 h-1 and 0.512 h, respectively; apparent clearance (CL/F) was 30.3 L/h (relative standard error [RSE] ± 8.25%) with an interindividual variability of 39.8% and interoccasion variability of 38.0%; meanwhile, apparent clearance of distribution (Q/F) was 17.0 L/h (RSE ± 12.1%) with and interindividual variability of 53.2%. The covariate analysis identified creatinine clearance (ClCrea) as an individual factor influencing the Cl of CsA. The predictive capacity of the population model was demonstrated to be effective since predictions made for new patients were accurate for C1 and C2 (MPPEs below 50%). Bayesian forecasting improved significantly in the second and third occasions. CONCLUSION: A population pharmacokinetic model was developed to reasonably estimate the individual cyclosporine clearance for patients. Hence, it can be utilized to individualize CsA doses for prompt and adequate achievement of target blood concentrations of CsA.

Sensitive, wide-range and high-throughput quantification of cyclosporine in whole blood using ultra-performance liquid chromatography coupled to tandem mass spectrometry and comparison with an antibody-conjugated magnetic immunoassay.

Because either trough or peak concentration at 2 h after administration is measured in routine therapeutic drug monitoring for cyclosporine A (CyA), a quantification method with a wide-range calibration curve capable of simultaneously measuring both concentrations is required. We developed a sensitive, wide-range and high-throughput quantification method for CyA in whole blood using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and compared patients' blood CyA levels measured by UPLC-MS/MS and antibody-conjugated magnetic immunoassay (ACMIA). Whole blood samples were prepared by solid-phase extraction using Oasis HLB µElution plate. The UPLC-MS/MS assay showed excellent linearity over a wide calibration range of 5-2500 ng/mL. Within-batch accuracy and precision as well as batch-to-batch accuracy and precision fulfilled the criteria of US Food and Drug Administration guidelines. The blood CyA concentrations measured by the UPLC-MS/MS assay correlated strongly with those measured by ACMIA. A Bland-Altman plot showed a fixed error between CyA concentrations measured by the two methods, and the concentrations measured by the UPLC-MS/MS method were consistently lower than those measured by ACMIA. We have succeeded to develop a sensitive, wide-range and high-throughput quantification method for CyA in whole blood using UPLC-MS/MS.

Simultaneous quantification of cyclosporin, tacrolimus, sirolimus and everolimus in whole blood by UHPLC-MS/MS for therapeutic drug monitoring.

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.

Calcineurin inhibitor-associated new-onset diabetes mellitus in chronic kidney disease treatment: a 4-year single-center cross-sectional study in China.

PURPOSE: To identify the risk factors of calcineurin inhibitor (CNI)-associated new-onset diabetes mellitus (NODM) in chronic kidney disease (CKD) treatment. METHODS: We retrospectively screened patients treated with CNIs in our hospital from January 2015 to December 2018. The inclusion criteria were as follows: a clear diagnosis of CKD and patients receiving CNI treatment. We compared patients with and without CNI-associated NODM. RESULTS: Ninety-eight of the 336 assessed patients met the inclusion criteria, 15 (15.3% [15/98]) of whom developed CNI-associated NODM. Multiple logistic regression analysis revealed that baseline glycosylated hemoglobin (OR=4.141; 1.024-16.743; p=0.046) and CNI trough concentration (1 year) (OR=1.028; 1.009-1.047, p=0.004) were independent risk factors for NODM. In contrast, glucocorticoid type (prednisone) (OR=0.075; 0.011-0.526, p=0.009) was identified as an independent protective factor for NODM. Using a receiver operating characteristic curve, a cutoff cyclosporin A trough concentration of 102.1 ng/mL was identified as a predictive factor of NODM. Univariate logistic regression showed that the incidence of diabetes was significantly higher in patients with baseline glycosylated hemoglobin in non-diabetic range but higher than 5.65% (10.2% vs. 29.2%, p=0.038). One NODM patient (6.7% [1/15]) recovered at 12.7 months after the onset of diabetes mellitus. CONCLUSIONS: We recommend that more attention be paid to patients with baseline glycosylated hemoglobin in non-diabetic range but higher than 5.65% during CKD treatment with CNIs. High trough concentrations of cyclosporin A, particularly those >102.1 ng/mL, contribute to NODM. CNI-associated NODM may be reversible in the treatment of CKD.

Clinical nephrotoxicity induced by cyclosporin A combined with hormone therapy for nephrotic syndrome.

This study aims to explore the nephrotoxicity due to use of combination of cyclosporine A and hormone in the treatment of nephrotic syndrome. From January 2018 to November 2019, 100 patients with primary nephrotic syndrome were divided into experimental and control groups, with 50 patients per group. The experimental group took oral cyclosporine A and prednisone tablets, while the control group received oral cyclosporine A combined with shock therapy. The contents of white blood cells, triglycerides, urine protein and cholesterol in the experimental group were lower than those in the control group, while their albumin content was significantly higher than the control values. Blood concentrations of cyclosporine A were significantly lower in non-nephrotoxic patients than in nephrotoxic patients. The high blood cyclosporine A level in patients (>200ng/mL) may be a factor for inducement of nephrotoxicity. Basal serum creatinine levels in nephrotoxic patients were significantly higher than those in non-nephrotoxic patients. Therefore, high basal creatinine level may be a contributing factor to nephrotoxicity. The combination of cyclosporine A and hormone is effective in the treatment of nephrotic syndrome. Blood cyclosporine A levels greater than 200ng/ml or elevated basal serum creatinine may be the cause of nephrotoxicity.

Food and lipid intake alters the pharmacokinetics of cyclosporine in kidney transplants.

Prevention of kidney graft rejection with cyclosporine leads to a large interindividual pharmacokinetic variability. However, food intake is likely to alter cyclosporine pharmacokinetics, and therefore its efficacy. The aim of our study was to evaluate the influence of food and lipid intake on cyclosporine pharmacokinetics. Twenty-four kidney grafted patients treated with Neoral® were included in this prospective monocentric study. In all patients, the pharmacokinetics of cyclosporine was evaluated in two occasions, after meal ('feed') and without meal ('fasting'). At each occasion, blood samples were collected at trough, and 0.5, 1, 2, 3, and 4 h after administration. Cyclosporine pharmacokinetics was described using a Bayesian pharmacokinetic model including two-compartments with first-order transfer and elimination rate constants, and a gamma absorption model. Influence of meal or olive oil, very common in Morocco, was tested as covariates on interoccasion variability parameters. Cyclosporine concentration-time data were satisfactorily described using the Bayesian pharmacokinetic model. Food intake significantly increased volume of distribution and decreased elimination of cyclosporine. The influence of oil intake explained a large part of this effect, suggesting that lipid intake was the main factor of pharmacokinetic variability due to food. This intake resulted in a decrease in area under the concentration curve between two administrations of 14.6%. Food, and especially lipid intake is likely to decrease the exposure to cyclosporine and may therefore lead to a decrease in treatment efficacy. Therefore, to ensure optimal immunosuppression in time, meal composition should remain as steady as possible.

Establishment of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Immunosuppressant Levels in the Peripheral Blood Mononuclear Cells of Chinese Renal Transplant Recipients.

BACKGROUND: Monitoring immunosuppressant levels, such as mycophenolic acid (MPA), cyclosporin A (CsA), and tacrolimus (TAC), in peripheral blood mononuclear cells (PBMCs) could be useful in organ transplant patients administered individualized therapy. The authors developed a liquid chromatography-tandem mass spectrometry assay technique to simultaneously determine immunosuppressant levels in PBMCs and assess their pharmacokinetics in Chinese renal allograft recipients. METHODS: PBMCs were isolated from the whole blood of 27 Chinese renal transplant patients using Ficoll-Paque Plus solution, and cell number was determined; acetonitrile treatment for protein precipitation, and gradient elution was performed on an Agilent Eclipse XDB-C18 column (3.5 µm, 2.1 × 100 mm) with mobile phase: water and methanol (containing 2 mM ammonium formate); flow rate: 0.3 mL·min. RESULTS: The calibration curves of MPA, CsA, and TAC had a linear range (ng·mL): 0.098-39.2 (r = 0.9987), 0.255-102 (r = 0.9969), and 0.028-11.2 (r = 0.9993), respectively. The extraction effects, matrix effects, and mean relative recovery of these immunosuppressants were 70.4%-93.2%, 72.7%-96.5%, and 90.1%-112.4%, respectively. The within-day and between-day coefficients of variation were <15%. The AUC0-12 of MPA in PBMCs correlated well with those in plasma. The level of MPA, CsA, and TAC in PBMCs might be more stable during dosing interval. CONCLUSIONS: The derived liquid chromatography-tandem mass spectrometry assay is suitable for simultaneously monitoring different immunosuppressants in PBMCs. Pharmacokinetic of MPA, CsA, and TAC displayed considerable interindividual variability. Intracellular monitoring of immunosuppressants may facilitate individualized therapy for renal allograft recipients.

Salting Out-Assisted Liquid-Liquid Extraction for Liquid Chromatography-Tandem Mass Spectrometry Measurement of Tacrolimus, Sirolimus, Everolimus, and Cyclosporine a in Whole Blood.

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.

Increased plasma concentrations of an antidyslipidemic drug pemafibrate co-administered with rifampicin or cyclosporine A in cynomolgus monkeys genotyped for the organic anion transporting polypeptide 1B1.

In vitro permeability and in vivo pharmacokinetics of pemafibrate were investigated in human intestinal and animal models untreated or pretreated with cyclosporine A or rifampicin to evaluate any drug interactions. Ratios of basal to apical apparent permeability (Papp) over apical to basal Papp in the presence of pH gradients decreased from 0.37 to 0.080 on rifampicin co-incubation, suggesting active transport of pemafibrate from basal to apical sides in intestinal models. Plasma concentrations of intravenously administered pemafibrate were enhanced moderately in control mice but only marginally in humanized-liver mice by oral pretreatment with rifampicin [an organic anion transporting polypeptide (OATP) 1B1 inhibitor] 1 h before the administration of pemafibrate. In three cynomolgus monkeys genotyped as wild-type OATP1B1 (2 homozygous and 1 heterozygous), oral dosing of cyclosporine A 4 h or rifampicin 1 h before pemafibrate administration significantly increased the areas under the plasma concentration-time curves (AUC) of intravenously administered pemafibrate by 4.9- and 7.4-fold, respectively. Plasma AUC values of three pemafibrate metabolites in cynomolgus monkeys were also increased by cyclosporine A or rifampicin. These results suggested that pemafibrate was actively uptaken in livers and rapidly cleared from plasma in cynomolgus monkeys; this rapid clearance was suppressible by OATP1B1 inhibitors.

Higher Concentrations of Cyclosporine Metabolites in Liver Transplant Recipients With a History of Viral and Bacterial Infections.

BACKGROUND: Infection remains a serious clinical problem in liver transplant (LTX) recipients. A higher risk of infection is connected with immunosuppression therapy. The aim of the study was to assess the relationships between infections' incidence and concentrations of cyclosporine (CsA) metabolites after LTX. METHODS: Forty-three liver transplant recipients receiving CsA were included in the study. With the use of liquid chromatography combined with tandem mass spectrometry, concentrations of CsA and its metabolites were measured: dihydroxylated cyclosporine metabolites (DiHCsA), trihydroxylated cyclosporine metabolites (TriHCsA), demethylcarboxylated cyclosporine metabolites (DemCarbCsA), AM1, AM9, and AM4N. The study protocol conformed with the Declaration of Helsinki. RESULTS: Patients with a history of Epstein-Barr virus (EBV) infection had higher DiHCsA, TriHCsA, DemCarbCsA, AM1/CsA, DiHCsA/CsA, TriHCsA/CsA i DemCarbCsA/CsA in comparison with group without such infection (P = .049, P = .037, P = .006, P = .018, P = .005, P = .027, and P = .026, respectively). LTX recipients with a history of all viral infections had higher DiHCsA, TriHCsA, DiHCsA/CsA, TriHCsA/CsA than patients without viral infections (P = .013, P = .021, P = .013, and P = .048, respectively). Multivariable analysis showed that AM1, DiHCsA, TriHCsA, DemCarbCsA, AM4N/CsA had positively influence on the incidence of all viral infections (ß = 0.0302, P = .0328; ß = 0.0699, P = .0453; ß = 0.6781, P = .0382; ß = 0.6767, P = .0414; and ß = 0.8307, P = .0267, respectively). In multivariable analysis, patients with a history of all bacterial infections had higher AM1 and higher AM1/CsA in comparison with LTX recipients without such infections (ß = 0.0118, P = .0279; and ß = 0.0099, P = .036, respectively). CONCLUSION: In liver transplant recipients with a history of viral or bacterial infections higher concentrations of CsA metabolites were found. Possibly CsA metabolites could be used to assess the risk of infection in patients after liver transplantation. It should be confirmed in further investigations.

Population Pharmacokinetic Modeling of Cyclosporine Among Malaysian Renal Transplant Patients: An Evaluation of Methods to Handle Missing Doses in Conventional Drug-Monitoring Data.

Cyclosporine is a primary drug in transplant immunosuppression regimens. It has a narrow therapeutic index and variable pharmacokinetic behavior. This study aimed to develop a population pharmacokinetic model of cyclosporine in Malaysian renal transplant recipients as well as to evaluate the performances of different methodsfor handling missing doses. A total of 2804 concentrationts predose and 2 hours after doses were collected retrospectively from 113 renal transplant patients on cyclosporine in Penang General Hospital. Model structure and pharmacokinetic parameters were estimated using nonlinear mixed-effects modeling software. Missing doses were handled using different methods to evaluate their performance. Covariate analysis was performed using stepwise forward addition (P < .05) followed by backward elimination (P < .001). Prediction-corrected visual predictive check and sampling-importance resampling methods were used to validate the final model. A 1-compartment model with first-order absorption and elimination best fitted the data. All methods to handle missing doses performed well with the missing dose method being superior to other methods and thus was applied in the final model. Cyclosporine clearance (CL/F) was estimated as 15.1 L/h, and volume of distribution (V/F) was 108 L. Postoperative time, sex, and calcium channel blockers were identified as significant covariates on CL/F, whereas sex and cholesterol level were identified as significant covariates on V/F. This is the first population pharmacokinetic model developed in Malaysian renal transplant patients using a large sample with an evaluation of different methods to handle missing doses in less informative conventional therapeutic drug-monitoring data.

Toll-Like 4 Receptor Expression on Peripheral Blood Mononuclear Cells in Renal Transplant Recipients Can Help to Indicate the Risk of Graft Deterioration in Patients Who Experienced an Episode of Symptomatic Cytomegalovirus Infection.

Data binding the expression of Toll-like 4 receptor (TLR4), transplanted kidney (KT) function, and symptomatic CMV infection (CMV+) are scarcely available. OBJECTIVE: To investigate the relationship between TLR4 expression (TLR4ex) in patients who had a relapse of CMV and transplant function. MATERIALS AND METHODS: TLR4ex was measured in peripheral blood mononuclear cells of KT recipients. We compared TLR4ex among 30 CMV+ patients and 87 patients without CMV infection (CMVneg). At the beginning (day 0) TLR4ex, as well as concentrations of cyclosporin A and tacrolimus were determined. All patients, CMV+ and CMVneg patients were divided according to the respective median of TLR4ex into groups of low-TLR4 expression (L-TLR4ex) and high-TLR4 expression (H-TLR4ex). Estimated glomerular filtration rate (EGFR) was assessed on day 0 and after the follow-up (F-up). The magnitudes of EGFR change (ΔEGFR) were evaluated. Stable treatment along the F-up period (median 11.9 months) was applied. RESULTS: TLR4ex of CMV+ in 67% was below median for all patients. For day 0, in CMV+: no link of TLR4ex with EGFR was found; TLR4ex was lower but day 0 EGFR did not differ from H-TLR4ex. In CMVneg, a GFR-TLR4ex link was present. Post F-up. In CMV+ with L-TLR4ex, EGFR declined, with no change in H-TLR4ex. In CMVneg with H-TLR4ex, EGFR increased, with no change in L-TLR4ex. Both regression and receiver operating characteristic curve analyses points out the impact of CMV+ and TLR4ex on eGFR and ΔEGFR. CONCLUSION: In CMV+, low TLR4ex increases the risk of EGFR deterioration. In CMVneg, high TLR4ex raises the chance of EGFR improvement.

Analysis of Immunosuppressant Drugs in Whole Blood by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

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.

Novel coronavirus (SARS-CoV-2) infection in a renal transplant recipient: Case report.

An ongoing outbreak of pneumonia associated with the severe acute respiratory coronavirus 2 (SARS-CoV-2) started in Wuhan, China, with cases now confirmed in multiple countries. The clinical course of patients remains to be fully characterized, clinical presentation ranges from asymptomatic infection to acute respiratory distress syndrome and acute renal failure, and no pharmacological therapies of proven efficacy yet exist. We report a case of SARS-CoV-2 infection in a renal transplant recipient with excellent outcome. This case states the importance of close monitoring of the concentration of cyclosporine in patients treated with lopinavir/ritonavir; the routine treatment of corticosteroid can be continued. This is a rare report of SARS-CoV-2 infection in a renal transplant recipient. Further data are needed to achieve better understanding of the impact of immunosuppressive therapy on the clinical presentation, severity, and outcome of SARS-CoV-2 infections in solid organ transplant recipients.

Simultaneous Determination of Everolimus, Sirolimus, Tacrolimus, and Cyclosporine-A by Mass Spectrometry.

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 [r2] > 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.

Using ISO/TS 20914:2019 to calculate the measurement uncertainty of immunosuppressive drugs in a clinical laboratory.

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).

Estimating Efflux Transporter-Mediated Disposition of Molecules beyond the Rule of Five (bRo5) Using Transporter Gene Knockout Rats.

Transporter gene knockout models are a practical and widely used tool for pharmacokinetic studies in drug discovery. P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are major efflux transporters that control absorption and bioavailability, and are important when determining oral drug disposition. To the best of our knowledge, beyond the rule of five (bRo5) molecules launched on the market to date tend to be substrates for efflux transporters. The purpose of this study is to evaluate in vivo the impact of efflux transporters on the oral absorption process and systemic clearance using rats which lack P-gp and/or Bcrp expression. We administered five bRo5 substrates (asunaprevir, cyclosporine, danoprevir, ledipasvir, and simeprevir) intravenously or orally to wild-type and Mdr1a, Bcrp, and Mdr1a/Bcrp knockout rats, calculated the clearance, oral bioavailability, and absorption rate profile of each substrate, and compared the results. Systemic clearance of the substrates in knockout rats changed within approximately ±40% compared to wild-types, suggesting the efflux transporters do not have a significant influence on clearance in rats. On the other hand, the oral absorption of substrates in the knockout rats, especially those lacking Mdr1a, increased greatly-between 2- and 5-fold more than in wild-types. This suggests that rat efflux transporters, especially P-gp, greatly reduce the oral exposure of these substrates. Moreover, results on the absorption rate-time profile suggest that efflux transporters are constantly active during the absorption period in rats. Transporter knockout rats are a useful in vivo tool for estimating the transporter-mediated disposition of bRo5 molecules in drug discovery.

Posttransplant Calcineurin Inhibitors Levels and Intrapatient Variability Are Not Associated With Long-term Outcomes Following Liver Transplantation.

BACKGROUND: There is an interest in understanding the association between early calcineurin inhibitors exposure post-liver transplantation (LT) and long-term outcomes. We aimed to analyze this association exploring median calcineurin inhibitor levels and intrapatient variability (IPV) in a multicenter, retrospective cohort. METHODS: Tacrolimus (Tac) and Cyclosporine (CsA) levels obtained during the first 15 days post-LT were collected. High immunosuppression (IS) was considered as a median of Tac, CsA blood trough levels 12 hours after drug administration, or blood levels 2 hours after drug administration higher than 10, 250, or 1200 ng/mL, respectively, or a peak of Tac >20 ng/mL. Optimal IS was defined as a median of Tac, CsA blood trough levels 12 hours after drug administration, or blood levels 2 hours after drug administration levels between 7 and 10, 150 and 250, or 800 and 1200 ng/mL. Low IS was defined as below the thresholds of optimal IS. IPV was estimated during the first 15 days post-LT. RESULTS: The study included 432 patients with a median follow-up of 8.65 years. IS regimen was based on either Tac or CsA in 243 (56.3%) and 189 (43.8%), respectively. There were no differences in terms of graft loss among low versus optimal and high IS groups (P = 0.812 and P = 0.451) nor in high versus low IPV (P = 0.835). Only viral hepatitis and arterial hypertension were independently associated with higher graft loss (hazard ratio = 1.729, P = 0.029 and hazard ratio = 1.570, P = 0.021). CONCLUSIONS: In contrast to what has previously been reported, no association was found between very early postoperative over IS or high IPV and long-term outcome measures following LT. Strategies aimed at reducing these long-term events should likely focus on other factors or on a different IS time window.