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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

Methotrexate pharmacokinetic is influenced by co-administration of cyclosporin in rheumatoid arthritis patients. Results from a randomized clinical trial.

The aim was to investigate if the pharmacokinetics of methotrexate (MTX) are affected by the addition of cyclosporin (CsA). Forty patients diagnosed with early rheumatoid arthritis (RA) were included in this open prospective study: 20 patients were treated with a dose of 7.5 mg MTX and a dose of 2.5 mg/kg CsA, 20 patients were treated with a dose of 7.5 mg MTX and placebo. Baseline measurements of plasma MTX and erythrocyte MTX were made. Area under the plasma concentration versus time curve (AUC) and other pharmacokinetic variables were estimated by means of a population based software model. Clinical improvement of 20-50-70% according to the American College of Rheumatology (ACR) and adverse events were evaluated ongoing for 52 weeks. We found that mean peak plasma MTX concentration was significantly higher in the MTX + CsA combination treatment group (p = .003). No differences in AUC, erythrocyte MTX or other pharmacokinetic parameters were found between the two treatment groups. Estimated Glomerular Filtration Rate (eGFR) decreased significantly in the MTX + CsA treatment group (p < .001), but no serious adverse events occurred in either of the two groups. In conclusion, CsA added to methotrexate treatment in early RA significantly increased peak-plasma MTX concentration, but other pharmacokinetic parameters and measurements of MTX were unchanged.

Inhibition of ABCB1 and ABCG2 at the Mouse Blood-Brain Barrier with Marketed Drugs To Improve Brain Delivery of the Model ABCB1/ABCG2 Substrate [11C]erlotinib.

P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are two efflux transporters at the blood-brain barrier (BBB), which effectively restrict brain distribution of diverse drugs, such as tyrosine kinase inhibitors. There is a crucial need for pharmacological ABCB1 and ABCG2 inhibition protocols for a more effective treatment of brain diseases. In the present study, seven marketed drugs (osimertinib, erlotinib, nilotinib, imatinib, lapatinib, pazopanib, and cyclosporine A) and one nonmarketed drug (tariquidar), with known in vitro ABCB1/ABCG2 inhibitory properties, were screened for their inhibitory potency at the BBB in vivo. Positron emission tomography (PET) using the model ABCB1/ABCG2 substrate [11C]erlotinib was performed in mice. Tested inhibitors were administered as i.v. bolus injections at 30 min before the start of the PET scan, followed by a continuous i.v. infusion for the duration of the PET scan. Five of the tested drugs increased total distribution volume of [11C]erlotinib in the brain ( VT,brain) compared to vehicle-treated animals (tariquidar, + 69%; erlotinib, + 19% and +23% for the 21.5 mg/kg and the 43 mg/kg dose, respectively; imatinib, + 22%; lapatinib, + 25%; and cyclosporine A, + 49%). For all drugs, increases in [11C]erlotinib brain distribution were lower than in Abcb1a/b(-/-)Abcg2(-/-) mice (+149%), which suggested that only partial ABCB1/ABCG2 inhibition was reached at the mouse BBB. The plasma concentrations of the tested drugs at the time of the PET scan were higher than clinically achievable plasma concentrations. Some of the tested drugs led to significant increases in blood radioactivity concentrations measured at the end of the PET scan (erlotinib, + 103% and +113% for the 21.5 mg/kg and the 43 mg/kg dose, respectively; imatinib, + 125%; and cyclosporine A, + 101%), which was most likely caused by decreased hepatobiliary excretion of radioactivity. Taken together, our data suggest that some marketed tyrosine kinase inhibitors may be repurposed to inhibit ABCB1 and ABCG2 at the BBB. From a clinical perspective, moderate increases in brain delivery despite the administration of high i.v. doses as well as peripheral drug-drug interactions due to transporter inhibition in clearance organs question the translatability of this concept.

Comparison of the effect of direct-acting antiviral with and without ribavirin on cyclosporine and tacrolimus clearance values: results from the ANRS CO23 CUPILT cohort.

PURPOSE: Direct-acting antiviral agents have demonstrated their efficacy in treating HCV recurrence after liver transplantation and particularly the sofosbuvir/daclatasvir combination. Pharmacokinetic data on both calcineurin inhibitors and direct-acting antiviral exposure in liver transplant recipients remain sparse. METHODS: Patients were enrolled from the ANRS CO23 CUPILT cohort. All patients treated with sofosbuvir/daclatasvir with or without ribavirin were included in this study when blood samples were available to estimate the clearance of immunosuppressive therapy before direct-acting antiviral initiation and during follow-up. Apparent tacrolimus and cyclosporine clearances were estimated from trough concentrations measured using validated quality control assays. RESULTS: Sixty-seven mainly male patients (79%) were included, with a mean age of 57 years and mean MELD score of 8.2; 50 were on tacrolimus, 17 on cyclosporine. Ribavirin was combined with sofosbuvir/daclatasvir in 52% of patients. Cyclosporine clearance remained unchanged as well as tacrolimus clearance under the ribavirin-free regimen. Tacrolimus clearance increased 4 weeks after direct-acting antivirals and ribavirin initiation versus baseline (geometric mean ratio 1.81; 90% CI 1.30-2.52). Patients under ribavirin had a significantly higher fibrosis stage (> 2) (p = 0.02) and lower haemoglobin during direct-acting antiviral treatment (p = 0.02) which impacted tacrolimus measurements. Direct-acting antiviral exposure was within the expected range. CONCLUSION: Our study demonstrated that liver transplant patients with a recurrence of hepatitis C who are initiating ribavirin combined with a sofosbuvir-daclatasvir direct-acting antiviral regimen may be at risk of lower tacrolimus concentrations because of probable ribavirin-induced anaemia and higher fibrosis score, although there are no effects on cyclosporine levels. TRIAL REGISTRATION: NCT01944527.

Indoxyl Sulfate Upregulates Liver P-Glycoprotein Expression and Activity through Aryl Hydrocarbon Receptor Signaling.

In patients with CKD, not only renal but also, nonrenal clearance of drugs is altered. Uremic toxins could modify the expression and/or activity of drug transporters in the liver. We tested whether the uremic toxin indoxyl sulfate (IS), an endogenous ligand of the transcription factor aryl hydrocarbon receptor, could change the expression of the following liver transporters involved in drug clearance: SLC10A1, SLC22A1, SLC22A7, SLC47A1, SLCO1B1, SLCO1B3, SLCO2B1, ABCB1, ABCB11, ABCC2, ABCC3, ABCC4, ABCC6, and ABCG2 We showed that IS increases the expression and activity of the efflux transporter P-glycoprotein (P-gp) encoded by ABCB1 in human hepatoma cells (HepG2) without modifying the expression of the other transporters. This effect depended on the aryl hydrocarbon receptor pathway. Presence of human albumin at physiologic concentration in the culture medium did not abolish the effect of IS. In two mouse models of CKD, the decline in renal function associated with the accumulation of IS in serum and the specific upregulation of Abcb1a in the liver. Additionally, among 109 heart or kidney transplant recipients with CKD, those with higher serum levels of IS needed higher doses of cyclosporin, a P-gp substrate, to obtain the cyclosporin target blood concentration. This need associated with serum levels of IS independent of renal function. These findings suggest that increased activity of P-gp could be responsible for increased hepatic cyclosporin clearance. Altogether, these results suggest that uremic toxins, such as IS, through effects on drug transporters, may modify the nonrenal clearance of drugs in patients with CKD.

Early isolated optic neuropathy caused by cyclosporine.

PURPOSE: To examine, for the first time, whether cyclosporine intake has an early isolated effect on the optic nerve. MATERIALS AND METHODS: This observational case series consisted of 192 eyes of 98 patients treated with cyclosporine. Patient age and duration and dosage of cyclosporine were recorded, and visual acuity, optic nerve function, visual fields, and visual evoked potential (VEP) were tested. Fundus examination was also performed. Patients with glaucoma, vascular retinopathies, and deep amblyopia were excluded. RESULTS: Mean patient age was 46 years, average duration of treatment was 6 years, and median dosage of cyclosporine was 200 mg daily. VEP was tested in 73 patients (142 eyes) and yielded a delayed P100 wave in 9 (12.32%) (14 eyes). Among these 9 patients, abnormal findings were also noted on the Ishihara colour test in 42.86% of the eyes, and on the visual field test in 64.3% of the eyes. Abnormal VEP showed a significant correlation (p < 0.05) with older age (> 46 years) and a non-significant correlation with longer duration of treatment. Higher abnormal VEP potential was not correlated with higher cyclosporine dose, and there was no correlation between abnormal VEP and blood level of cyclosporine. CONCLUSION: Optic neuropathy was significantly associated with older age in cyclosporine-treated patients. A correlation between optic neuropathy with longer duration of cyclosporine treatment was noted but was not statistically significant. We suggest that tests of optic nerve function, including VEP, be a part of the follow-up of patients receiving cyclosporine.

Co-administration of cyclosporine and ticagrelor may lead to a higher exposure to cyclosporine: a case report of a 49-year-old man.

ADVERSE EVENT: A drug interaction leading to higher exposure to cyclosporine. DRUGS IMPLICATED: Cyclosporine and ticagrelor. THE PATIENT: A 49-year-old man with a stable renal graft, managed with cyclosporine with stable trough blood concentrations for several years, was treated with ticagrelor for unstable angina pectoris. EVIDENCE THAT LINKS THE DRUG TO THE EVENT: The timeline was consistent with the appearance of an interaction, the interaction was confirmed by an increase in trough concentration of cyclosporine, and there were no alternative causes that by themselves could have caused the increase in cyclosporine exposure. MANAGEMENT: Cessation of ticagrelor. MECHANISM: Inhibition of CYP3A4 and P-glycoprotein by ticagrelor. IMPLICATIONS FOR THERAPY: Clinicians should be aware of this potential interaction as ticagrelor is frequently prescribed in individuals using cyclosporine. Close monitoring of cyclosporine serum concentrations is warranted to avoid overdosing of cyclosporine. A pharmacokinetic study is needed to further examine the probable interaction between cyclosporine and ticagrelor.

Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry for Cyclosporine Analysis in Human Whole Blood and Comparison With an Antibody-Conjugated Magnetic Immunoassay.

BACKGROUND: Various immunoassays have been used for cyclosporine A (CsA) analysis in human whole blood; however, they could not fully satisfy the requirements of criteria for accuracy and specificity in CsA measurement. The liquid chromatography tandem mass spectrometry is a gold method for CsA analysis. The aim of the study was to develop and validate an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for CsA analysis and establish its agreement with an antibody-conjugated magnetic immunoassay (ACMIA) in clinical sample analysis. METHODS: An UHPLC-MS/MS method for CsA analysis in human whole blood was developed, validated, and applied in 85 samples, which were also tested by ACMIA. The agreement between UHPLC-MS/MS and ACMIA was evaluated by Bland-Altman plot. RESULTS: The calibration range was 5-2000 ng/mL. The inaccuracy and imprecision were -4.60% to 5.56% and less than 8.57%, respectively. The internal standard-normalized recovery and matrix factor were 100.4%-110.5% and 93.5%-107.6%, respectively. The measurements of ACMIA and UHPLC-MS/MS were strongly correlated (r > 0.98). Evaluated by Bland-Altman plot, the 95% limit of agreement of the ACMIA:UHPLC-MS/MS ratio was 88.7%-165.6%, and the mean bias of the ratio was 21.1%. CONCLUSIONS: A rapid, simple, accurate, and reliable UHPLC-MS/MS method for CsA analysis in human whole blood was developed, validated, and applied in 85 samples. On average, 21.1% overestimation was observed in ACMIA compared with that in the UHPLC-MS/MS. Further and larger studies are required to identify whether this degree of variance could be accepted by clinicians.

Measurement uncertainty and metrological traceability of whole blood cyclosporin A mass concentration results obtained by UHPLC-MS/MS.

BACKGROUND: Traceable and accurate results of cyclosporine A (CsA) mass concentrations in whole blood are required to ensure the monitoring of immunosuppressive therapy in transplant recipients. Metrological traceability and measurement uncertainty can allow ensuring reliability and comparability of these results over time and space. In this study, we provide a practical and detailed example of how the traceability and uncertainty of mass concentration of CsA results, obtained using an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) procedure, can be described and estimated. METHODS: Traceability was described mainly according to ISO 17511 and information obtained from certificates facilitated with the manufacturer's calibrators. Uncertainty estimation was performed using the bottom-up and top-down approaches. For the bottom-up approach, the most relevant sources of uncertainty were identified and later used to estimate the standard, combined and expanded uncertainties. For the top-down approach, expanded uncertainty was estimated directly using intralab quality control data mainly. RESULTS: Mass concentration of CsA results was traceable to the manufacturer's product calibrators used to calibrate the UHPLC-MS/MS procedure. The expanded uncertainties estimated by the bottom-up and top-down approaches were 7.4% and 7.2%, respectively. CONCLUSIONS: After performing the bottom-up and top-down approaches, we observed that their results were quite similar. This fact would confirm that the top-down approach could be sufficient for estimating uncertainty of CsA mass concentrations in whole blood results in clinical laboratories. Finally, we hope that this study can help and motivate clinical laboratories to describe metrological traceability and to perform measurement uncertainty studies based on the simpler top-down approach.

Converting cyclosporine A from intravenous to oral administration in hematopoietic stem cell transplant recipients and the role of azole antifungals.

PURPOSE: Cyclosporine A (CsA) is the most widely used immunosuppressive agent after a hematopoietic stem cell transplantation (HSCT). Although recommendations for CsA dose conversion from intravenous to oral administration differ from 1:1 to 1:3, most studies did not consider the role of azole antifungals as an important confounder. Therefore, we assess the optimal conversion rate of CsA from intravenous to oral administration in HSCT recipients, taking into account the concomitant use of azole antifungals. METHODS: We retrospectively included patients from a large database of 483 patients who underwent a HSCT and received intravenous CsA as part of the conditioning regimen and peritransplant immunosuppression. All patients were converted from intravenous to oral administration in a 1:1 conversion rate. We collected for each patient three CsA trough concentrations during intravenous and oral administration, directly before and after conversion to oral administration. RESULTS: We included 71 patients; 50 patients co-treated with fluconazole, 10 with voriconazole, and 11 without azole co-medication. In patients with voriconazole, the dose-corrected CsA concentration (CsA concentration divided by CsA dosage) was not different between intravenous and oral administration (2.6% difference, p = 0.754), suggesting a CsA oral bioavailability of nearly 100%. In patients with fluconazole and without azole co-medication, the dose-corrected CsA concentration was respectively 21.5% (p < 0.001) and 25.2% (p = 0.069) lower during oral administration. CONCLUSIONS: In patients with voriconazole, CsA should be converted 1:1 from intravenous to oral administration. In patients with fluconazole and without azole co-medication, a 1:1.3 substitution is advised to prevent subtherapeutic CsA concentrations.