Association of adalimumab trough concentrations and treatment response in patients with juvenile idiopathic arthritis.

OBJECTIVE: The objective of this study was to assess the relationship between adalimumab trough concentrations and treatment response in paediatric patients with JIA. METHODS: This was a monocentric cohort study of JIA patients treated with adalimumab. Clinical data and samples were collected during routine follow-up. Adalimumab trough concentrations were quantified by a novel liquid chromatography-tandem mass spectrometry assay. Anti-adalimumab antibodies were measured in samples with trough concentrations of ≤5mg/l. Disease activity was evaluated using the clinical Juvenile Arthritis DAS with 71-joint count (cJADAS71). Response to adalimumab was defined according to recent international treat-to-target guidelines. RESULTS: A total of 35 adalimumab trough samples were available from 34 paediatric patients with JIA. Although there was no significant difference in adalimumab dose, trough concentrations were significantly lower in patients with secondary failure [median 1.0 mg/l; interquartile range (IQR) 1.0-5.3] compared with patients with primary failure (median 13.97 mg/l; IQR 11.81-16.67) or an adequate response (median 14.94 mg/l; IQR 10.31-16.19) to adalimumab. CONCLUSION: Adalimumab trough concentrations were significantly lower in JIA patients with secondary failure compared with patients with primary failure or an adequate response to adalimumab. Our results suggest that trough concentration measurements could identify JIA patients who require increased adalimumab doses to achieve or maintain therapeutic drug concentrations.

Influence of allopurinol on thiopurine associated toxicity: A retrospective population-based cohort study.

AIMS: Thiopurines are important for treating inflammatory bowel disease, but are often discontinued due to adverse effects. Concomitant use of allopurinol might lower the risk of these unwanted effects, but large studies in the general population are lacking. The aims of this study were to evaluate rates of hepatotoxicity, myelotoxicity, pancreas toxicity and therapy persistence in adult thiopurine users with or without allopurinol. METHODS: A retrospective population-based cohort study was conducted within current thiopurine users (Clinical Practice Research Datalink). Among these patients, co-use of allopurinol was compared to non-use. Hazard ratios (HRs) for hepatotoxicity, myelotoxicity and pancreatitis were derived using time-dependent Cox proportional hazards models, and were adjusted for potential confounders. Persistence of thiopurine use was evaluated using Log-rank statistics. RESULTS: Patients using thiopurines (n = 37 360) were identified of which 1077 were concomitantly taking allopurinol. A 58% decreased risk of hepatotoxicity was observed in those concomitantly taking allopurinol (HR 0.42; 95% CI 0.30-0.60; NNT 46). Rate of myelotoxicity (HR 0.96; 95% CI 0.89-1.03) was not influenced. Risk of pancreatitis was increased (HR 3.00; 95% CI 1.01-8.93; NNH 337), but was only seen in those with active gout (suggesting confounding by indication). Finally, allopurinol co-users were able to maintain thiopurine therapy over twice as long as those not on allopurinol (3.9 years vs. 1.8 years, P < 0.0001). CONCLUSION: In thiopurine users, allopurinol is associated with a 58% reduced risk of hepatotoxicity. In addition, thiopurine persistence was prolonged by 2.1 years in allopurinol users. These data support the use of allopurinol in individuals requiring thiopurine therapy.

Comparison between coagulation factor VIII quantified with one-stage activity assay and with mass spectrometry in haemophilia A patients: Proof of principle.

INTRODUCTION: Haemophilia A is a hereditary bleeding disorder caused by a factor VIII (FVIII) deficiency. As biomarker, FVIII activity is used to classify disease severity and to monitor treatment. The one-stage clotting assay (OSA) is performed to measure FVIII activity, but OSA's limitations may result in misclassification of disease severity or suboptimal monitoring of treatment. Measurement of FVIII plasma concentration with liquid chromatography-tandem mass spectrometry (LC-MS/MS) might overcome these challenges. The objective is to investigate the correlation between FVIII activity and concentration, and determinants for differences between the two methods. METHODS: In this cross-sectional study, all haemophilia A patients receiving standard-of-care were eligible for inclusion. Within the activity categories of <1 IU/dL, 1-5 IU/dL, >5-40 IU/dL, >40-150 IU/dL and >150-600 IU/dL, we randomly selected 15-20 plasma samples and compared FVIII concentration (LC-MS/MS) to FVIII activity (OSA) with linear regression and Bland-Altman analysis. Potential determinants for differences were analysed with linear regression. RESULTS: Inclusion was 87 samples. Bland-Altman analysis demonstrated an overall mean difference of -1% with an SD of 64% between the two methods. Large differences were correlated with the presence of anti-FVIII antibodies (133% [95% CI: 81, 185] n = 5) and use of exogenous FVIII products (-37% [95% CI: -65,-9] n = 58), for example plasma-derived and B-domain-modified FVIII products. CONCLUSIONS: Despite good overall correlation between the two methods, relative differences were large, especially for samples with anti-FVIII antibodies or exogenous FVIII products. These differences may have clinical impact. More research is needed to determine the value of FVIII plasma concentration in comparison with FVIII activity.

High efavirenz levels but not neurofilament light plasma levels are associated with poor neurocognitive functioning in asymptomatic HIV patients.

The aim of this study is to assess the effect of efavirenz exposure on neurocognitive functioning and investigate plasma neurofilament light (Nfl) as a biomarker for neurocognitive damage. Sub-analysis of the ESCAPE-study, a randomised controlled trial where virologically suppressed, cognitively asymptomatic HIV patients were randomised (2:1) to switch to rilpivirine or continue on efavirenz. At baseline and week 12, patients underwent an extensive neuropsychological assessment (NPA), and serum efavirenz concentration and plasma Nfl levels were measured. Subgroups of elevated (≥ 4.0 mg/L) and therapeutic (0.74 to< 4.0 mg/L) baseline efavirenz concentration were made. Differences between these groups in baseline NPA Z-scores and in delta scores after efavirenz discontinuation were assessed. Nfl level was measured using an ELISA analysis using single molecule array (Simoa) technology. Correlation of plasma NFL with NPA Z-scores was evaluated using a linear mixed model. The elevated group consisted of 6 patients and the therapeutic group of 48. At baseline, the elevated group showed lower composite Z-scores (median - 1.03; IQR 0.87 versus 0.27; 0.79. p 0.02). This effect was also seen on the subdomains verbal (p 0.01), executive functioning (p 0.02), attention (p < 0.01) and speed (p 0.01). In the switch group, the elevated group improved more on composite scores after discontinuing efavirenz (mean 0.58; SD 0.32 versus 0.22; 0.54, p 0.15). No association between plasma Nfl and composite Z-score was found. High efavirenz exposure is associated with worse cognitive functioning compared with patients with therapeutic concentrations. Plasma Nfl is not a suitable biomarker to measure cognitive damage in this group.

Exposure Variability and Target Attainment of Vancomycin: A Systematic Review Comparing Intermittent and Continuous Infusion.

BACKGROUND: Studies comparing the clinical outcomes between vancomycin intermittent infusion (InI) and continuous infusion (CoI) treated patients are generally underpowered. Moreover, due to large differences in the design and efficacy end points in these studies, a meta-analysis of the currently available data is not feasible. Therefore, this systematic review aimed to compare the exposure variability and target attainment with vancomycin during InI and CoI. PATIENTS AND METHODS: A literature search was performed, and clinical studies reporting on vancomycin-treated populations were selected. After exclusion of reviews, case reports, and articles not published in the English language, 505 articles were screened for reported data on vancomycin serum concentrations. A total of 34 studies were included in the review. Relative standard deviations reported in the included studies were assessed, and vancomycin serum concentration variability and target attainment were compared between vancomycin InI and CoI. RESULTS: The variability in serum concentrations was significantly larger for InI than for CoI (relative standard deviations 46.5% and 32.1%, respectively; P = 0.001). Notably, variability appeared to be independent of the study population or design. Studies directly comparing target attainment between both modes of administration denoted higher and faster target attainment with CoI in all instances. CONCLUSIONS: In conclusion, CoI was associated with lower variabilities in the serum concentration and favorable target attainment rates compared with InI. These findings are important because vancomycin exposure is considered a major predictor of the patients' clinical outcomes. However, the role of lower serum concentration variability and higher target attainment rates in achieving better clinical outcomes needs to be evaluated in patients treated with vancomycin CoI compared with InI.

A semi-mechanistic model based on glutathione depletion to describe intra-individual reduction in busulfan clearance.

AIM: To develop a semi-mechanistic model, based on glutathione depletion and predict a previously identified intra-individual reduction in busulfan clearance to aid in more precise dosing. METHODS: Busulfan concentration data, measured as part of regular care for allogeneic hematopoietic cell transplantation (HCT) patients, were used to develop a semi-mechanistic model and compare it to a previously developed empirical model. The latter included an empirically estimated time effect, where the semi-mechanistic model included theoretical glutathione depletion. As older age has been related to lower glutathione levels, this was tested as a covariate in the semi-mechanistic model. Lastly, a therapeutic drug monitoring (TDM) simulation was performed comparing the two models in target attainment. RESULTS: In both models, a similar clearance decrease of 7% (range -82% to 44%), with a proportionality to busulfan metabolism, was found. After 40 years of age, the time effect increased with 4% per year of age (0.6-8%, P = 0.009), causing the effect to increase more than a 2-fold over the observed age-range (0-73 years). Compared to the empirical model, the final semi-mechanistic model increased target attainment from 74% to 76%, mainly through better predictions for adult patients. CONCLUSION: These results suggest that the time-dependent decrease in busulfan clearance may be related to gluthathione depletion. This effect increased with older age (>40 years) and was proportional to busulfan metabolism. The newly constructed semi-mechanistic model could be used to further improve TDM-guided exposure target attainment of busulfan in patients undergoing HCT.

Does Circadian Rhythm Affect the Pharmacokinetics of Once-Daily Tobramycin in Adults With Cystic Fibrosis?

BACKGROUND: In the era of multiple daily dosing of systemic aminoglycosides, a circadian rhythm in the clearance of these vital antibiotics has been demonstrated in animals and healthy volunteers. Over the past decade, once-daily dosing regimens have been proved to be less nephrotoxic and were therefore adopted worldwide for most indications requiring treatment with an aminoglycoside. In this study, the effect of the time of administration on the pharmacokinetics of once-daily tobramycin in adults with cystic fibrosis (CF) experiencing a pulmonary exacerbation was investigated. METHODS: In this open randomized study, patients with CF received intravenous tobramycin at 8:00 or 22:00 hours. Pharmacokinetic and kidney function parameters were compared between the 2 groups. RESULTS: Twenty-five patients were included. The mean weight-corrected clearances of tobramycin were 1.46 versus 1.43 mL/h*kg (P = 0.50) and mean volumes of distribution were 0.25 versus 0.27 L/kg (P = 0.54) for the 8:00 and 22:00 groups, respectively. In addition, no significant differences were detected in changes in estimated clearances of creatinine or tobramycin on day 1 and day 8 in the 8:00 or 22:00 group, indicating that there was no decline in clearance over time. At day 8 of therapy, the increase in serum blood urea nitrogen in the 22:00 group was significantly higher than that in the 8:00 group (1.8 versus 0.2 mmol/L, P = 0.015). CONCLUSIONS: The time of administration (8:00 versus 22:00) did not affect tobramycin pharmacokinetics in the adult CF population studied. The increase in serum blood urea nitrogen in the 22:00 group requires further investigation.

Quantification of T Cell Binding Polyclonal Rabbit Anti-thymocyte Globulin in Human Plasma with Liquid Chromatography Tandem-Mass Spectrometry.

The addition of rabbit anti-human thymocyte globulin (ATG) to the conditioning regimen prior to allogeneic hematopoietic cell transplantation has significantly reduced the risk of graft-versus-host disease (GvHD) and graft failure. However, ATG has a small therapeutic window. Overexposure of ATG post-HCT hampers T cell immune reconstitution and has been associated with increased relapse rates and viral reactivations, whereas underexposure has been associated with an increased incidence of GvHD, both of which lead to increased mortality. Therapeutic drug monitoring of T cell binding ATG plasma levels provides a means to optimize dosing for patients at high risk for graft failure to ensure timely T cell immune reconstitution and subsequently increase survival chances. This manuscript describes the first liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to quantify the pharmacologically active fraction of polyclonal ATG in plasma. This was achieved through immunoaffinity purification of active ATG from plasma with Jurkat T cells. After the binding and washing, samples were eluted, denatured, and trypsin-digested. Signature peptides originating from the IgG constant chain were measured with LC-MS/MS. Critical method parameters were optimized, and the method was successfully validated following European Medicines Agency (EMA) guidelines. The method covered the therapeutic range of ATG and was validated at a lower limit of quantification (LLOQ) of 1 AU/mL with an overall CV and bias of 11.8% and - 2.5%, respectively. In conclusion, we developed a LC-MS/MS-based method to quantify active polyclonal rabbit ATG in human plasma. We suggest that this novel assay can be used to monitor and optimize dosing of ATG in clinical practice.

Clinical Trial Simulation To Optimize Trial Design for Fludarabine Dosing Strategies in Allogeneic Hematopoietic Cell Transplantation.

Optimal fludarabine exposure has been associated with improved treatment outcome in allogeneic hematopoietic cell transplantation, suggesting potential benefit of individualized dosing. A randomized controlled trial (RCT) comparing alternative fludarabine dosing strategies to current practice may be warranted, but should be sufficiently powered for a relevant end point, while still feasible to enroll. To find the optimal design, we simulated RCTs comparing current practice (160 mg/m2 ) to either covariate-based or therapeutic drug monitoring (TDM)-guided dosing with potential outcomes being nonrelapse mortality (NRM), graft failure, or relapse, and ultimately overall survival (covering all three aforementioned outcomes). The inclusion in each treatment arm (n) required to achieve 80% power was calculated for all combinations of end points and dosing comparisons. The trial requiring the lowest n for sufficient power compared TDM-guided dosing to current practice with NRM as primary outcome (n = 70, NRM decreasing from 21% to 5.7%). We conclude that a superiority trial is feasible.

Unbound Plasma, Total Plasma, and Whole-Blood Tacrolimus Pharmacokinetics Early After Thoracic Organ Transplantation.

BACKGROUND AND OBJECTIVE: Therapeutic drug monitoring of tacrolimus whole-blood concentrations is standard care in thoracic organ transplantation. Nevertheless, toxicity may appear with alleged therapeutic concentrations possibly related to variability in unbound concentrations. However, pharmacokinetic data on unbound concentrations are not available. The objective of this study was to quantify the pharmacokinetics of whole-blood, total, and unbound plasma tacrolimus in patients early after heart and lung transplantation. METHODS: Twelve-hour tacrolimus whole-blood, total, and unbound plasma concentrations of 30 thoracic organ recipients were analyzed with high-performance liquid chromatography-tandem mass spectrometry directly after transplantation. Pharmacokinetic modeling was performed using non-linear mixed-effects modeling. RESULTS: Plasma concentration was < 1% of the whole-blood concentration. Maximum binding capacity of erythrocytes was directly proportional to hematocrit and estimated at 2700 pg/mL (95% confidence interval 1750-3835) with a dissociation constant of 0.142 pg/mL (95% confidence interval 0.087-0.195). The inter-individual variability in the binding constants was considerable (27% maximum binding capacity, and 29% for the linear binding constant of plasma). CONCLUSIONS: Tacrolimus association with erythrocytes was high and suggested a non-linear distribution at high concentrations. Monitoring hematocrit-corrected whole-blood tacrolimus concentrations might improve clinical outcomes in clinically unstable thoracic organ transplants. CLINICAL TRIAL REGISTRATION: NTR 3912/EudraCT 2012-001909-24.

Clinical Pharmacokinetics and Impact of Hematocrit on Monitoring and Dosing of Tacrolimus Early After Heart and Lung Transplantation.

The calcineurin inhibitor tacrolimus is an effective immunosuppressant and is extensively used in solid organ transplantation. In the first week after heart and lung transplantation, tacrolimus dosing is difficult due to considerable physiological changes because of clinical instability, and toxicity often occurs, even when tacrolimus concentrations are within the therapeutic range. The physiological and pharmacokinetic changes are outlined. Excessive variability in bioavailability may lead to higher interoccasion (dose-to-dose) variability than interindividual variability of pharmacokinetic parameters. Intravenous tacrolimus dosing may circumvent this high variability in bioavailability. Moreover, the interpretation of whole-blood concentrations is discussed. The unbound concentration is related to hematocrit, and changes in hematocrit may increase toxicity, even within the therapeutic range of whole-blood concentrations. Therefore, in clinically unstable patients with varying hematocrit, aiming at the lower therapeutic level is recommended and tacrolimus personalized dosing based on hematocrit-corrected whole-blood concentrations may be used to control the unbound tacrolimus plasma concentrations and subsequently reduce toxicity.

High Variability of Whole-Blood Tacrolimus Pharmacokinetics Early After Thoracic Organ Transplantation.

BACKGROUND AND OBJECTIVE: Oral tacrolimus is initiated perioperatively in heart and lung transplantation patients. There have been few studies on oral tacrolimus pharmacokinetics early post-transplantation, even though tacrolimus-related toxicity may occur early, potentially leading to morbidity and mortality. Therefore, we aimed to study the pharmacokinetics of oral tacrolimus in thoracic organ recipients during the first days after transplantation. METHODS: We conducted a pharmacokinetic study in 30 thoracic organ transplants at intensive care at the University Medical Center Utrecht in the first week post-transplantation. Twelve-hour whole-blood tacrolimus profiles were examined using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and analysed via population pharmacokinetic modelling. RESULTS: The concentration-time profiles showed high variability. Concentrations at 12 h were outside the target range in 69% of the cases. A two-compartment model with mixed first-order and zero-order absorption adequately described tacrolimus concentrations. The typical value of the apparent clearance was 19.6 L/h (95% CI 16.2-22.9), and the apparent distribution volumes of central and peripheral compartments, V1 and V2, were 231 L (95% CI 199-267) and 521 L (95% CI 441-634), respectively. Inter-occasion (dose-to-dose) variability far exceeded the interindividual variability (IIV), with an estimated variability in relative bioavailability of 55% (95% CI 48.5-64.4). CONCLUSIONS: The high variability of tacrolimus pharmacokinetics early after thoracic organ transplantation is largely due to excessive variability in bioavailability, making individualised dosing based on measured concentrations futile. To bypass this bioavailability issue, we suggest administering tacrolimus intravenously and aiming below the upper therapeutic range early post-transplantation. Clinical Trial Registraion: NTR 3912/EudraCT 2012-001909-24.

Simultaneous Quantification of Free Adalimumab and Infliximab in Human Plasma Using a Target-Based Sample Purification and Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring of tumor necrosis factor alpha (TNF-α) inhibitors such as adalimumab (ADM) and infliximab (IFX) is considered of added value for patients with systemic inflammatory diseases. In contrast to enzyme-linked immunosorbent assay methods, liquid chromatography-tandem mass spectrometry methods allow for simultaneous quantification of multiple target antibodies in 1 run and thus providing a higher sample throughput. We describe a fast sample work-up strategy for the absolute and simultaneous quantification of ADM and IFX therapeutic monoclonal antibodies in human plasma samples using a target-specific sample purification in combination with liquid chromatography-tandem mass spectrometry. METHODS: The sample purification was based on the selective capture of ADM and IFX in human plasma or serum using biotinylated TNF-α (b-TNF-α), which was coated on a streptavidin 96-well plate. After elution, analytes were heat denatured and trypsin digested to obtain signature peptides for quantification. Stable isotopically labeled ADM and IFX were introduced as internal standard before sample purification. RESULTS: The method was successfully validated following current European medicines agency guidelines. The linear dynamic rage for both analytes were 1-32 mcg/mL with an excellent mean coefficient of determination, R = 0.9994 for ADM and 0.9996 for IFX. Within-run and between-run imprecision and accuracy were within acceptance criteria. Cross-validation against enzyme-linked immunosorbent assay method showed a high between-method correlation R = 0.962 for ADM and R = 0.982 for IFX. CONCLUSIONS: This method provides an easy, efficient, and cost-effective workflow for therapeutic drug monitoring patients treated with ADM or IFX.

Quantification of coagulation factor VIII in human plasma with liquid chromatography tandem mass spectrometry using a selective sample purification with camelid nanobodies.

Patients with hemophilia A are currently diagnosed and monitored by measuring the activity of coagulation factor VIII (FVIII) in plasma mostly with the one-stage clotting assay (OSA). Although the OSA is routinely available in many clinical laboratories, it has in some circumstances relatively low sensitivity and specificity. Therefore, the FVIII activity as a biomarker does not always correlate with the bleeding phenotype. Therefore, we have developed a liquid chromatography tandem mass spectrometry method to quantify the concentration of coagulation FVIII in plasma which would allow us to investigate the relation between FVIII plasma concentration, FVIII activity and bleeding tendency in future studies. LC-MS/MS method was set up by firstly dissociation Von Willebrand factor (VWF) from coagulation factor VIII by triggering the coagulation cascade to occur thus generating active factor VIII (FVIIIa). FVIIIa was then selectively extracted by means of immunoaffinity interaction using anti-FVIII camelid nanobody, after which FVIIIa was eluted, heat denatured and trypsin digested. Finally, a FVIII specific peptide was used as a surrogate for quantification by mass spectrometry. Critical method parameters such as antibody amount, incubation time, sample volume and type of streptavidin 96 well plate were optimized. The method was validated according to European Medicines Agency (EMA) guidelines where an LLOQ of 1 ng/mL was obtained using 50 µL of citrate plasma sample. Within-run and between-run accuracy and precision for quality control (QC) samples, LLOQ (1 ng/mL), QC Low (5 ng/mL), QC Med (150 ng/mL), QC High (300 ng/mL) were within the threshold of 15% relative standard deviation (RSD) and Bias. The selective immunoaffinity method which was used in combination with a highly sensitive mass spectrometer allowed for an unpresented LLOQ of 1 ng/mL utilizing 50 µL plasma sample. This method will be used to investigate the beneficial value of FVIII plasma concentration which may be used in conjunction with FVIII activity for patient diagnosis and dosage optimization.

Six-step workflow for the quantification of therapeutic monoclonal antibodies in biological matrices with liquid chromatography mass spectrometry - A tutorial.

The promising pipeline of therapeutic monoclonal antibodies (mAbs) demands robust bioanalytical methods with swift development times for pharmacokinetic studies. Over the past decades ligand binding assays were the methods of choice for absolute quantification. However, the production of the required anti-idiotypic antibodies and ligands limits high-throughput method development for sensitive, accurate, and reproducible quantification of therapeutic mAbs. In recent years, high-resolution liquid chromatography tandem mass-spectrometry (LC-MS) systems have enabled absolute quantification of therapeutic mAbs with short method development times. These systems have additional benefits, such as a large linear dynamic range, a high specificity and the option of multiplexing. Here, we briefly discuss the current strategies for the quantification of therapeutic mAbs in biological matrices using LC-MS analysis based on top-down and middle-down quantitative proteomics. Then, we present the widely used bottom-up method in a six-step workflow, which can be used as guidance for quantitative LC-MS/MS method development of mAbs. Finally, strengths and weaknesses of the bottom-up method, which currently provides the most benefits, are discussed in detail.

Nonlinear protein binding of phenytoin in clinical practice: Development and validation of a mechanistic prediction model.

AIMS: To individualize treatment, phenytoin doses are adjusted based on free concentrations, either measured or calculated from total concentrations. As a mechanistic protein binding model may more accurately reflect the protein binding of phenytoin than the empirical Winter-Tozer equation that is routinely used for calculation of free concentrations, we aimed to develop and validate a mechanistic phenytoin protein binding model. METHODS: Data were extracted from routine clinical practice. A mechanistic drug protein binding model was developed using nonlinear mixed effects modelling in a development dataset. The predictive performance of the mechanistic model was then compared with the performance of the Winter-Tozer equation in 5 external datasets. RESULTS: We found that in the clinically relevant concentration range, phenytoin protein binding is not only affected by serum albumin concentrations and presence of severe renal dysfunction, but is also concentration dependent. Furthermore, the developed mechanistic model outperformed the Winter-Tozer equation in 4 out of 5 datasets in predicting free concentrations in various populations. CONCLUSIONS: Clinicians should be aware that the free fraction changes when phenytoin exposure changes. A mechanistic binding model may facilitate prediction of free phenytoin concentrations from total concentrations, for example for dose individualization in the clinic.

Unbound Fraction of Clozapine Significantly Decreases with Elevated Plasma Concentrations of the Inflammatory Acute-Phase Protein Alpha-1-Acid Glycoprotein.

BACKGROUND: During inflammation, elevated total (unbound plus protein-bound) clozapine plasma concentrations have been observed. Elevated alpha-1-acid glycoprotein concentrations during inflammation are suggested to cause increased plasma clozapine-alpha-1-acid glycoprotein binding, resulting in elevated total clozapine plasma concentrations without significant changes in unbound concentrations. Here, we investigated the association between alpha-1-acid glycoprotein plasma concentrations and clozapine unbound fraction. METHODS: First, 25 and 60 µL of alpha-1-acid glycoprotein solution (20 mg/mL) were added to plasma samples (n = 3) of clozapine users (spiking experiment). Second, the association between alpha-1-acid glycoprotein plasma concentration and clozapine unbound fraction was assessed in patient samples (patient study). Samples were determined by liquid chromatography-tandem mass spectrometry. Data were analyzed with a paired t test (spiking experiment) and an unpaired t test (patient study). RESULTS: The spiking experiment showed significantly lower mean unbound fractions following 25- and 60-µL alpha-1-acid glycoprotein spikes (relative reductions of 28.3%, p = 0.032 and 43.4%, p = 0.048). In the patient study, total clozapine plasma concentrations were 10% higher in elevated (n = 6) compared with normal alpha-1-acid glycoprotein (n = 20) samples [525 µg/L vs. 479 µg/L, mean difference = 47 µg/L (95% confidence interval -217 to 310), p = 0.72]. Elevated alpha-1-acid glycoprotein samples had a 26% lower mean unbound fraction compared with normal samples [1.22% vs. 1.65%, mean difference = -0.43% (95% confidence interval -0.816 to -0.0443), p = 0.03]. CONCLUSIONS: Both the spiking experiment and patient study showed a significant association between elevated alpha-1-acid glycoprotein plasma concentrations and a lower clozapine unbound fraction. Future studies should include clinical data to examine whether this association is clinically relevant, suggesting any clozapine dose adjustments.

Pharmacokinetics and safety of tobramycin nebulization with the I-neb and PARI-LC Plus in children with cystic fibrosis: A randomized, crossover study.

AIMS: We aimed to compare the pharmacokinetics (PK) and safety profile of tobramycin inhalation solution (TIS) using the I-neb device to the standard PARI-LC Plus nebulizer in children with cystic fibrosis. METHODS: A randomized, open-label, crossover study was performed. In 2 separate study visits, blood samples from 22 children were collected following TIS nebulization with I-neb (75 mg) and PARI-LC Plus (300 mg). Study visits were separated by 1 month, in which 1 of the study nebulizers was used twice daily. Tobramycin PK for both nebulizers was established using measured tobramycin concentrations and Bayesian PK modelling software. Hearing and renal function tests were performed to test for aminoglycoside associated toxicity. In addition to standard estimated glomerular filtration rate values, biomarkers for tubular injury (KIM-1 and NAG) were measured. Patient and nebulizer satisfaction were assessed. RESULTS: Inhalations were well tolerated and serum trough concentrations below the predefined toxic limit were reached with no significant differences in PK parameters between nebulizers. Results of audiometry and estimated glomerular filtration rate revealed no abnormalities. However, increased urinary NAG/creatinine ratios at visit 2 for both nebulizers suggest TIS-induced subclinical tubular kidney injury. Nebulization time was 50% shorter and patient satisfaction was significantly higher with the I-neb. CONCLUSIONS: Nebulization of 75 mg TIS with the I-neb in children with cystic fibrosis resulted in comparable systemic exposure to 300 mg TIS with the PARI-LC Plus and was well tolerated and preferred over the PARI-LC Plus. Long-term safety of TIS nebulization should be monitored clinically, especially regarding the effects on tubular kidney injury.