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Non-specific binding in in vitro metabolism systems leads to an underestimation of the true intrinsic metabolic clearance of compounds being studied. Therefore in vitro binding needs to be accounted for when extrapolating in vitro data to predict the in vivo metabolic clearance of a compound. While techniques exist for experimentally determining the fraction of a compound unbound in in vitro metabolism systems, early in drug discovery programmes computational approaches are often used to estimate the binding in the in vitro system. Experimental fraction unbound data (n = 60) were generated in liver microsomes (fumic) from five commonly used pre-clinical species (rat, mouse, dog, minipig, monkey) and humans. Unbound fraction in incubations with mouse, rat or human hepatocytes was determined for the same 60 compounds. These data were analysed to determine the relationship between experimentally determined binding in the different matrices and across different species. In hepatocytes there was a good correlation between fraction unbound in human and rat (r2=0.86) or mouse (r2=0.82) hepatocytes. Similar correlations were observed between binding in human liver microsomes and microsomes from rat, mouse, dog, Göttingen minipig or monkey liver microsomes (r2 of >0.89, n = 51 − 52 measurements in different species). Physicochemical parameters (logP, pKa and logD) were predicted for all evaluated compounds. In addition, logP and/or logD were measured for a subset of compounds. Binding to human hepatocytes predicted using 5 different methods was compared to the measured data for a set of 59 compounds. The best methods evaluated used measured microsomal binding in human liver microsomes to predict hepatocyte binding. The collated physicochemical data were used to predict the human fumic using four different in silico models for a set of 53–60 compounds. The correlation (r2) and root mean square error between predicted and observed microsomal binding was 0.69 & 0.20, 0.47 & 0.23, 0.56 & 0.21 and 0.54 & 0.26 for the Turner-Simcyp, Austin, Hallifax-Houston and Poulin models, respectively. These analyses were extended to include measured literature values for binding in human liver microsomes for a larger set of compounds (n=697). For the larger dataset of compounds, microsomal binding was well predicted for neutral compounds (r2=0.67 − 0.70) using the Poulin, Austin, or Turner-Simcyp methods but not for acidic or basic compounds (r2<0.5) using any of the models. While the lipophilicity-based models can be used, the in vitro binding should be measured for compounds where more certainty is needed, using appropriately calibrated assays and possibly established weak, moderate, and strong binders as reference compounds to allow comparison across databases. [ABSTRACT FROM AUTHOR]

Clinical and genetic influencing factors on free fraction of mycophenolic acid (MPA) have rarely been discussed. The present study investigated whether the clinical and genetic factors could explain the variability in the pharmacokinetics of free MPA (fMPA) and total MPA (tMPA) in Chinese paediatric and adult renal transplant recipients. Twenty‐eight paediatric and 31 adult patients were enrolled, and the concentrations of tMPA and fMPA were determined at 0 h (predose) and 0.5, 1, 1.5, 2, 4, 5, 8, 9, 10 and 12 h after mycophenolate mofetil administration. Genetic polymorphisms of UGTs (rs671448, rs1042597, rs2741049, rs62298861, rs7439366, rs12233719) and ABCC2 (rs717620) were simultaneously determined. The clinical and genetic data were analysed and reported. tMPA and fMPA concentrations adjusted for dose per body weight were consistently higher in adults than in paediatric patients. In the paediatric group, only albumin and time after transplantation correlated significantly with the MPA‐free fraction variation, which could explain 32.4% of the variability. Besides, ABCC2 polymorphism, albumin and time after transplantation correlated significantly with the MPA‐free fraction variation in adults, which could explain 56.9% of the variability. The influencing factors in the paediatric group are different from those in adults, which may be due to age‐related transporter expression. [ABSTRACT FROM AUTHOR]

The aim of this study is to establish and test an in vitro digestion-in situ absorption model that can mimic in vivo drug flux by employing a physiologically relevant value of the membrane surface area ( S )/volume ( V ) ratio for accurate prediction of oral drug absorption from lipid-based formulations (LBFs). Three different types of LBFs (Type IIIA-MC, Type IIIA-LC, and Type IV) loaded with cinnarizine (CNZ), a lipophilic weak base with borderline permeability, and a control suspension were prepared. Subsequently, a simultaneous in vitro digestion-permeation experiment was conducted using a side-by-side diffusion cell with a dialysis membrane having a low S / V value. During digestion, CNZ partially precipitated for Type IV, while it remained solubilized in the aqueous phase for Type IIIA-MC and Type IIIA-LC in the donor compartment. However, in vitro drug fluxes for Type IIIA-MC and Type IIIA-LC were lower than those for Type IV due to the reduced free fraction of CNZ in the donor compartment. In pharmacokinetic studies, a similar improvement in in vivo oral exposure relative to suspension was observed, regardless of the LBFs used. Consequently, a poor correlation was found between in vitro permeation and areas under the plasma concentration-time curve (AUC oral ) ( R = 0.087). A luminal concentration measurement study revealed that this discrepancy was attributed to the extremely high absorption rate of CNZ in the gastrointestinal tract compared to that across a dialysis membrane evaluated by the in vitro digestion-permeation model, i.e., the absorption of CNZ in vivo was completed regardless of the extent of the free fraction, owing to the rapid removal of CNZ from the intestine. Subsequently, we aimed to predict the oral absorption of CNZ from the same formulations using a model that demonstrated high drug flux by employing the physiologically relevant 2 = 0.087). A luminal concentration measurement study revealed that this discrepancy was attributed to the extremely high absorption rate of CNZ in the gastrointestinal tract compared to that across a dialysis membrane evaluated by the in vitro digestion-permeation model, i.e., the absorption of CNZ in vivo was completed regardless of the extent of the free fraction, owing to the rapid removal of CNZ from the intestine. Subsequently, we aimed to predict the oral absorption of CNZ from the same formulations using a model that demonstrated high drug flux by employing the physiologically relevant S / V value and rat jejunum segment as an absorption sink (for replicating in vivo intestinal permeability). Predigested formulations were injected into the rat intestinal loop, and AUC loop values were calculated from the plasma concentration-time profiles. A better correlation was found between AUC loop and AUC oral ( R 2 = 0.72), although AUC loop underestimated AUC oral for Type IV due to the precipitation of CNZ during the predigestion process. However, this result indicated the importance of mimicking the in vivo drug absorption rate in the predictive model. The method presented herein is valuable for the development of LBFs.

Purpose: Cyclodextrin (CD) is commonly used to enhance the solubility of oral drugs. However, with the increase of CD concentrations, the fraction of free drug molecules decreases, which may potentially impede drug absorption. This study aims to predict the optimal ratio between drug and CD to achieve the best absorption efficiency by computational simulation. Methods: First, a physiologically based pharmacokinetic (PBPK) model was developed. This model can continuously adjust absorption according to free drug fraction and was validated against two model drugs, progesterone (PG) and andrographolide (AG). The further analysis involves 3-D surface graphs to investigate the relationship between free drug amount, theoretically absorbable concentration, and contents of drug and CD in the formulation. Results: The PBPK model predicted the PK behavior of two drugs well. The concentration ratio of drug to CD, leading to maximal free drug amount and the best absorption efficiency, is nearly the same as the slope determined in the phase solubility test. The new modified PBPK model and 3-D surface graph can easily predict the absorption difference of formulations with various drug/CD ratios. Conclusion: This PBPK model and 3-D surface graph can predict the absorption and determine the optimal concentration ratio of CD formulation, which could accelerate the R&D of CD formulation. [ABSTRACT FROM AUTHOR]

Most quantitative research methods are based on measuring either the total or the free concentration of an analyte in a sample. However, this is often insufficient for the study of complex biological systems. The main objective of this research was to develop new methods for providing more information from samples: the free concentration (C f), the total concentration (C t), and the plasma binding capacity (PBC). Samples were processed using microextraction and ultrafiltration. For each of these techniques, two quantification procedures were used: addition of isotopically labeled standard and repeated analysis of the same sample. The new methods were validated by analyzing clinical samples and samples with known concentrations. Methods based on addition of labeled compound were found to be the fastest, and most reproducible. For analysis of clinical samples, methods based on microextraction were more sensitive and more accurate than those based on ultrafiltration. For analysis of pooled plasma samples, the overall accuracy of all approaches to determine PBC , testosterone C f , and testosterone C t was between 94 and 109%, 87–113%, and 94–122% respectively. The new approach goes beyond a simple concentration measurement, giving more information from clinical samples, with great potential for personalizing drug dosage and therapy to the needs of individual patients. [ABSTRACT FROM AUTHOR]

A simple and sensitive LC–MS/MS method was established to quantify total and free mycophenolic acid (MPA) plasma concentrations during immunosuppressive medication for pediatric renal transplantation. The chromatographic separation was performed with the Hypersil GOLD C18 column, using a mobile phase consisting of 0.1% formic acid in water and acetonitrile (60:40, v/v) at an isocratic flow rate of 0.4 ml/min. An Agilent 6420 triple quadrupole mass spectrometer was operated via a positive electrospray ionization interface using the transitions m/z 321.14 → 206.9 for MPA and m/z 324.15 → 209.9 for MPA‐d3 (internal standard). The linearity was 0.1–50 μg/ml for total MPA and 0.0025–0.5 μg/ml for free MPA. The within‐run and between‐run precisions were all <5% and accuracy was within 96.23–107.63%. The validated method was successfully aspplied to a pharmacokinetic study in 28 pediatric renal recipients. The mean free fraction of MPA in our patients was 0.89% (ranging from 0.62 to 1.25%) and albumin level played a major role in the variability of free fraction of MPA, thus, in pediatric patients with hypoproteinemia, close free drug monitoring and dose adjustments should be considered to prevent toxicity. [ABSTRACT FROM AUTHOR]

A brief note on the studies we have conducted on total and free drug concentration of thousands of drug discovery compounds in HeLa cells as measured by an approach inspired by the work of Professor Per Arturrsson. We conclude that the familiar QSAR equations of Corwin Hansch which were modelled as a bell shape by using logP and -logP2 terms can be similarly seen in our results and this can be interpreted with the aid of chromatographic Immobilised Artificial Membrane measurements. We also point out the differences between our measurements and those widely used based on Artificial Membrane Permeability Assays. [ABSTRACT FROM AUTHOR]

Abstract The molecular composition and biomarker distribution of various occurrences of organic matter in argillaceous source rocks developed in fresh and saline lacustrine environments were revealed by successive treatments of solvent extraction followed by acid hydrolysis using gas chromatography–mass spectrometry. The free fraction obtained by solvent extraction provided abundant geochemical information concerning the sedimentary environment, thermal maturity and biogenic origin, and obvious differences existed between fresh and saline lacustrine source rock samples. Our research results indicate that the carbonate-mineral-bound (CM-bound) fraction released by successive acid hydrolysis could also serve as a significant biogenic indicator, as the bicyclic sesquiterpenoids, indicative of Botryococcus braunii origin, were specifically detected in quite high abundance in the acid-soluble fraction. In addition, the light end hydrocarbons were much better preserved in the acid-soluble fraction, and elemental sulfur was only detected in the CM-bound fraction, suggesting a relatively confined environment for the CM-bound fraction, which thus could preserve additional geochemical information compared to that of the free fraction. The CM-bound fraction also exhibited discernable differences between fresh and saline lacustrine samples. Therefore, it can be concluded that comprehensive analysis of free and CM-bound fractions in the argillaceous source rocks can provide a more authentic and objective interpretation of geologic conditions.

Determination of free drug fraction (f u) in plasma can be challenging for labile covalent modulators due to the off-target reactivity of chemical warheads to matrix proteins. The resulting poor drug recovery yields low confidence in f u. Two approaches using diluted plasma and low temperature (4 & 20 °C) for equilibrium dialysis (ED) have been investigated using covalent modulators including osimertinib, ibrutinib, rociletinib, afatinib, neratinib and acalabrutinib. Our data indicate that stability of covalent modulators in plasma varies in different species, and drug depletion may lead to overestimation of f u if true equilibrium is not reached. Additionally, although ED at low temperature improves the recovery of covalent modulators, the impact of low temperature may lead to underestimate of f u. Overall, ED using diluted plasma is a preferred method because of its faster equilibrium, improved recovery and free of temperature effect on f u. If low temperature ED must be used for extremely labile compounds, precaution must be taken to ensure no temperature dependence of f u in plasma. Nevertheless, an orthogonal ED approach is recommended for labile covalent modulators to confirm the true equilibrium and impact of temperature on f u. Additionally, this strategy can be used for determining f u of other liable compounds. [ABSTRACT FROM AUTHOR]

A population pharmacokinetic (PK) model was developed using data pooled from 6 clinical studies (3 in healthy volunteers and 3 in cancer patients) to characterize total and free plasma concentrations of erdafitinib following single‐ and multiple‐dose administration, to understand clinically relevant covariates, and to quantify the inter‐ and intraindividual variability in erdafitinib PK. An open, linear, 3‐compartment disposition model with first‐order absorption and a lag time was used to describe the PK profile of total and free erdafitinib plasma concentrations. The PK of erdafitinib were linear and time independent. After oral administration, erdafitinib was rapidly absorbed, with a time to maximum concentration between 2 and 4 hours. In patients, erdafitinib total apparent oral clearance was 0.200 L/h (median free fraction = 0.24%), and the effective terminal half‐life of total drug was 76.4 hours. Interindividual variability in PK parameters was moderate for oral clearance and central volume of distribution, and large for absorption rate and peripheral volume of distribution. Sex and renal function were significant covariates on free oral clearance, while weight, sex, and α1‐acid‐glycoprotein were significant on oral central volume of distribution. Age, race, and mild hepatic impairment were not significant covariates of erdafitinib exposure. Given that the magnitude of the covariate effects were within 25% of reference values and that the recommended dosing regimen of erdafitinib comprises individual dose up‐titrations and reductions based on presence or absence of toxicities, the clinical relevance of the investigated covariates is expected to be limited, and no dose adjustments are warranted. [ABSTRACT FROM AUTHOR]

Darunavir (DRV) is an HIV protease inhibitor commonly used as part of antiretroviral treatment regimens globally for children and adolescents. It requires a pharmacological booster, such as ritonavir (RTV) or cobicistat. To better understand the pharmacokinetics (PK) of DRV in this younger population and the importance of the RTV boosting effect, a population PK substudy was conducted within SMILE trial, where the maintenance of HIV suppression with once daily integrate inhibitor + darunavir/ritonavir in children and adolescents is evaluated. A joint population PK model that simultaneously used total DRV, unbound DRV, and total RTV concentrations was developed. Competitive and non-competitive models were examined to define RTV's influence on DRV pharmacokinetics. Linear and non-linear equations were tested to assess DRV protein binding. A total of 443 plasma samples from 152 adolescents were included in this analysis. Darunavir PK was best described by a one-compartment model first-order absorption and elimination. The influence of RTV on DRV pharmacokinetics was best characterized by ritonavir area under the curve on DRV clearance using a power function. The association of non-linear and linear equations was used to describe DRV protein binding to alpha-1 glycoprotein and albumin, respectively. In our population, simulations indicate that 86.8% of total and unbound DRV trough concentrations were above 0.55 mg/L [10 times protein binding-adjusted EC 50 for wild-type (WT) HIV-1] and 0.0243 mg/L (10 times EC 90 for WT HIV-1) targets, respectively. Predictions were also in agreement with observed outcomes from adults receiving 800/100 mg DRV/r once a day. Administration of 800/100 mg of DRV/r once daily provides satisfactory concentrations and exposures for adolescents aged 12 years and older., Competing Interests: The authors declare no conflict of interest.

There is an expanding number of applications for preclinical positron emission tomography (PET) imaging. Kinetic modeling of PET data provides rich multiparameter information on radiotracer uptake and binding in tissue from a single experiment. In this chapter, we provide a practical step-by-step protocol to assist with collection of PET data for kinetic modeling studies in rats and mice., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Patient-specific factors can alter the pharmacokinetic disposition of valproic acid. Specifically, the free fraction of valproic acid can increase substantially in patients with hypoalbuminemia or as serum drug concentrations rise due to saturable protein binding. Direct measurement of free serum drug concentrations allows for accurate assessment of drug levels, but the assay may not be readily available in all institutions. The effect of hypoalbuminemia on free fraction has been quantified and serves as the basis of an equation used to "correct" measured total valproic acid concentrations. The aim of this study was to evaluate the accuracy of the equation.Methods: This retrospective study included adult patients with measurable free and total valproic acid concentrations between July 2014 and June 2017. The primary aim was to assess the relationship between measured and predicted free valproic acid concentrations. Free levels were categorized as subtherapeutic, therapeutic, or supratherapeutic based on the reference range of 7-23 mg/L. Concordance was defined as measured and predicted concentrations falling within the same category.Results: The analysis included 174 patients with a median age of 58 years and a median albumin of 3 g/dL. The majority of patients were hospitalized (88.5%). Concordance occurred in 56.9% of samples. A Spearman's correlation coefficient of 0.60 (p < 0.001) was found between the measured and predicted free valproic acid concentrations. Concordance of concentrations was 42% for ICU patients, 63% for floor patients, and 65% for outpatients. Of those with discordant concentrations, 97% of the predicted concentrations underestimated the measured concentrations.Conclusions: There is discordance between predicted and measured free serum valproic acid concentrations when using the proposed equation. Because of the potential impact of underestimation and variability of free valproic acid concentrations, a measured free level is the ideal option for therapeutic drug monitoring of valproic acid. [ABSTRACT FROM AUTHOR]

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

Abstract Objectives The antibiotic teicoplanin, used for the treatment of infections caused by Gram-positive bacteria, is highly bound to plasma proteins (percentage protein binding, %PB, around 90%) and therapeutic plasma levels of total teicoplanin are 10–100 mg/L. Because of the low free concentrations (i.e. <1–10 mg/L), current immunoassays are not able to quantify free teicoplanin concentrations, although they might be more relevant in therapeutic drug monitoring than total concentrations. Design and methods In this study, an ultra-high performance liquid chromatography – high resolution mass spectrometry (UHPLC-HRMS) method for the quantification of total and free teicoplanin in K 2 EDTA plasma samples was developed and validated. Furthermore, %PB obtained by ultrafiltration was compared with that obtained by equilibrium dialysis using spiked samples from healthy subjects. Analytes were separated using a phenylhexyl column, gradient mobile phase analysis was used, total run time was 4.5 min and teicoplanin was detected by orbitrap MS. Results The precision and accuracy were below 15% and within ±15%, respectively and teicoplanin was found to be stable for at least 14 days in plasma at 4 °C. The %PB of teicoplanin in spiked plasma from healthy subjects as obtained by ultrafiltration (94.1 ± 1.3%) was in good agreement with that obtained by equilibrium dialysis (93.6 ± 0.4%), whereas mean %PB of teicoplanin in samples from infected patients who received the antibiotic was 87.7 ± 4.2% (range: 79.6–95.4%). Conclusion A novel highly sensitive UHPLC-HRMS method was developed and validated for the quantification of total and free teicoplanin in human K 2 EDTA plasma samples. Amongst others, this method is suitable for therapeutic drug monitoring. [ABSTRACT FROM AUTHOR]

The molecular composition and biomarker distribution of various occurrences of organic matter in argillaceous source rocks developed in fresh and saline lacustrine environments were revealed by successive treatments of solvent extraction followed by acid hydrolysis using gas chromatography-mass spectrometry. The free fraction obtained by solvent extraction provided abundant geochemical information concerning the sedimentary environment, thermal maturity and biogenic origin, and obvious differences existed between fresh and saline lacustrine source rock samples. Our research results indicate that the carbonate-mineral-bound (CM-bound) fraction released by successive acid hydrolysis could also serve as a significant biogenic indicator, as the bicyclic sesquiterpenoids, indicative of Botryococcus braunii origin, were specifically detected in quite high abundance in the acid-soluble fraction. In addition, the light end hydrocarbons were much better preserved in the acid-soluble fraction, and elemental sulfur was only detected in the CM-bound fraction, suggesting a relatively confined environment for the CM-bound fraction, which thus could preserve additional geochemical information compared to that of the free fraction. The CM-bound fraction also exhibited discernable differences between fresh and saline lacustrine samples. Therefore, it can be concluded that comprehensive analysis of free and CM-bound fractions in the argillaceous source rocks can provide a more authentic and objective interpretation of geologic conditions. [ABSTRACT FROM AUTHOR]

Unbound drug concentration in the brain would be the true exposure responsible for specific target occupancy. Drug exposures from preclinical are total concentrations of those over/underestimate the clinical dose projection. With the application of mass spectrometry, the current work proposes a definite measure of test drug exposures at serotonin-2A occupancy. The 5-HT2A occupancy of antagonist in the rat brain has determined with non-radiolabeled tracer MDL-100,907 at an optimized dose (3 µg/kg) and treatment time (30 min). Equilibrium dialysis method determines the in vitro free fraction of the test antagonist in untreated rat brain homogenates and plasma. Drug-free fractions derived the unbound concentration (EC50) in plasma and brain at test doses. The corresponding binding affinities (Ki) correlated with the unbound concentrations. Except for quetiapine, the ED50 values in the dose-occupancy curves of antagonists are close and ranged from 1 to 3 mg/kg. The test drug quetiapine, eplivanserin, and clozapine showed high free fractions in plasma, but for ketanserin and olanzapine, the brain free fraction was higher. The correlation between the unbound EC50 of the antagonists and corresponding Ki values was good (r2=0.828). The improved EC50 accuracy with unbound concentrations was 10-250 folds in plasma and 10-170 folds in the brain. Further, the free fractions (fu, plasma/fu, brain) of test drugs had shown a correlation of ∼83% with brain permeability (Ctotal brain/Ctotal plasma), a limiting factor. Thus, correlating the occupancy with unbound exposure and pharmacology would result in an accurate measurement of drug potency and optimizes in selecting the clinical dose. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate variables affecting the valproate (VPA) free fraction and develop an equation for computing free VPA concentration from total VPA concentration.Methods: Trough total and free VPA concentrations were collected from patients who participated in a prospective VPA monotherapy trial. All available paired data of trough total and free VPA concentrations were included. Significant variables from the univariate analysis were evaluated in a multivariate model.Results: A total of 902 concomitant total and free VPA concentrations were available. Multivariate analysis showed that total VPA concentration, age and gender were significantly associated with VPA free fraction. However, the effect size of total VPA concentration was substantially higher than that of gender and age. VPA free fraction remained stable at around 10% for total VPA concentration between 20 and 60 μg/mL with subsequent linear increases for higher concentration. A scatter plot correlating total and free VPA concentrations showed that a quadratic equation best fitted the data, accounting for 88% of the free VPA concentration variance.Conclusions: An increase in the total VPA concentration results in corresponding linear and non-linear rise in the VPA free fraction and free VPA concentration, respectively. The total daily dose of VPA should be increased in smaller increments whenever a total VPA concentration of 60 μg/mL is reached. When drug monitoring is needed, we recommend measuring the free VPA concentration. If this test is unavailable, and for patients with normal albumin levels, it can be predicted from the total VPA concentration using the generated equation. [ABSTRACT FROM AUTHOR]

This study describes the development and validation of a method for the analysis of unbound plasma concentrations of oxcarbazepine (OXC) and of the enantiomers of its active metabolite 10-hydroxycarbazepine (MHD) [S-(+)- and R-(−)-MHD] using liquid chromatography with tandem mass spectrometry (LC–MS/MS). Additionally, the free fraction of the drug is described in healthy volunteers (n = 12) after the oral administration of 300 mg OXC/12 h for 5 days. Plasma aliquots of 200 μL were submitted to ultrafiltration procedure and 50 μL of the ultrafiltrate were extracted with a mixture of tert -butyl methyl ether:dichloromethane (2:1, v/v). OXC and the MHD enantiomers were separated on a OD-H chiral phase column. The method was linear in the range of 4.0–2.0 μg/mL for OXC and of 20.0–6.0 μg/mL plasma for the MHD enantiomers. The limit of quantification was 4 ng for OXC and 20 ng for each MHD enantiomer/mL plasma. The intra- and inter-day precision and inaccuracy were less than 15%. The free fraction at the time of peak plasma concentration of OXC was 0.27 for OXC, 0.37 for S-(+)-MHD and 0.42 for R-(−)-MHD. Enantioselectivity in the free fraction of MHD was observed, with a higher proportion of R-(−)-MHD compared to S-(+)-MHD. [ABSTRACT FROM AUTHOR]

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

Study Objective Because the pharmacokinetic evaluation of valproic acid ( VPA) based on total drug concentration may be misleading in patients with hypoalbuminemia as a result of saturable protein binding and saturable metabolism, we sought to investigate the usefulness of therapeutic drug monitoring of unbound VPA concentration in a real-world clinical context, with a focus on clinically significant neurologic adverse outcomes. Design Retrospective analysis. Setting Large academic tertiary care hospital in Montreal, Canada. Patients Forty-one adults, hospitalized or followed as outpatients, for whom unbound VPA concentration testing was performed between January 1, 2008, and April 30, 2015. Patients were retrospectively identified by using the hospital's central laboratory database. Measurements and Main Results In the multiple linear regression analysis, the two variables that significantly predicted unbound VPA concentration were total VPA concentration (p<0.001) and albumin concentration (p<0.001). The correlation between total VPA concentration and the number of neurologic adverse symptoms was 0.187 (p=0.241), whereas the correlation between unbound VPA concentration and the number of neurologic adverse symptoms was 0.384 (p=0.013). The performance of total and unbound VPA concentrations in predicting the presence of at least one neurologic adverse symptom, as determined by the receiver operating characteristic curve, was 0.642 (95% confidence interval [ CI] 0.449-0.836, p=0.167) and 0.776 (95% CI 0.629-0.923, p=0.007), respectively. Conclusion This study showed that in the presence of hypoalbuminemia, high unbound VPA concentrations can be observed despite normal or low total VPA concentrations. It also demonstrated that high unbound VPA concentrations are associated with clinically significant neurologic adverse symptoms. Clinicians should be aware that unbound VPA concentration monitoring may be required in the presence of hypoalbuminemia. [ABSTRACT FROM AUTHOR]

Objective Given that only the free non-protein-bound concentration of an antiepileptic drug ( AED) crosses the blood-brain barrier, entering the brain and producing an antiepileptic effect, knowledge and measurement of the free drug fraction is important. Such data are sparse, particularly for newer AEDs, and have arisen from the use of disparate methodologies and settings over the past six decades. We report on the protein binding of 25 AEDs that are available for clinical use, along with two pharmacologically active metabolites (carbamazepine-epoxide and N-desmethyl clobazam), using standardized methodology and under set conditions. Methods The protein binding of the various AEDs was undertaken in sera of 278 patients with epilepsy. Separation of the free non-protein-bound component was achieved by using ultracentrifugation (Amicon Centrifree Micropartition System) under set conditions: 500 μl serum volume; centrifugation at 1,000 g for 15 min, and at 25°C. Free and total AED concentrations were measured by use of fully validated liquid chromatography/mass spectroscopy (LC/MS) techniques. Results Gabapentin and pregabalin are non-protein-bound, whereas highly bound AEDs (≥88%) include clobazam, clonazepam, perampanel, retigabine, stiripentol, tiagabine, and valproic acid as well as the N-desmethyl-clobazam (89%) metabolite. The minimally bound drugs (<22%) include ethosuximide (21.8%), lacosamide (14.0%), levetiracetam (3.4%), topiramate, (19.5%) and vigabatrin (17.1%). Ten of the 25 AEDs exhibit moderate protein binding (mean range 27.7-74.8%). Significance These data provide a comprehensive comparison of serum protein binding of all available AEDs including the metabolites, carbamazepine-epoxide and N-desmethyl-clobazam. Knowledge of the free fraction of these AEDs can be used to optimize epilepsy treatment. [ABSTRACT FROM AUTHOR]

Amplification of the gene encoding Myeloid cell leukemia-1 (Mcl-1) is one of the most common genetic aberrations in human cancer and is associated with high tumor grade and poor survival. Recently, we reported on the discovery of high affinity Mcl-1 inhibitors that elicit mechanism-based cell activity. These inhibitors are lipophilic and contain an acidic functionality which is a common chemical profile for compounds that bind to albumin in plasma. Indeed, these Mcl-1 inhibitors exhibited reduced in vitro cell activity in the presence of serum. Here we describe the structure of a lead Mcl-1 inhibitor when bound to Human Serum Albumin (HSA). Unlike many acidic lipophilic compounds that bind to drug site 1 or 2, we found that this Mcl-1 inhibitor binds predominantly to drug site 3. Site 3 of HSA may be able to accommodate larger, more rigid compounds that do not fit into the smaller drug site 1 or 2. Structural studies of molecules that bind to this third site may provide insight into how some higher molecular weight compounds bind to albumin and could be used to aid in the design of compounds with reduced albumin binding. [ABSTRACT FROM AUTHOR]