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Aims: Cardiopulmonary bypass (CPB) reduces the plasma protein‐binding rate of some anaesthetics and can enhance their pharmacological effects by increasing the unbound drug fraction. However, whether these changes occur with remifentanil remains to be explored. We investigated the changes in the protein‐binding rate of remifentanil during CPB compared with propofol. Methods: Thirteen patients (≥18 years old) who were scheduled to undergo cardiovascular surgery with CPB were included. Arterial blood samples were collected to measure the plasma concentrations of remifentanil and propofol before CPB (T1), 30 (T2) and 60 (T3) minutes after the start of CPB, and 30 min after CPB discontinuation (T4). The samples were immediately centrifuged to separate the plasma after blood collection. Equilibrium dialysis was used to separate the unbound fraction. The remifentanil and propofol concentrations were measured by liquid chromatography‐mass spectrometry. The protein‐binding rate was calculated based on the total and unbound fraction of each drug. Results: The remifentanil protein‐binding rates at each time point were 27.9% ± 11.2% (T1), 13.5% ± 4.4% (T2), 14.0% ± 3.3% (T3) and 24.5% ± 6.9% (T4). The propofol protein‐binding rates were 97.5% ± 0.7% (n = 4; T1), 95.8% ± 1.4% (T2), 95.3% ± 1.3% (T3) and 95.8% ± 1.1% (T4). The protein binding rates of both drugs decreased during CPB and reversed after CPB. The change in the unbound fraction was 1.2‐fold for remifentanil and 1.7‐1.9‐fold for propofol. Conclusions: Unlike propofol, remifentanil might not demonstrate significantly enhanced pharmacological effects during CPB. [ABSTRACT FROM AUTHOR]

Accurate prediction of drug-drug interactions (DDI) from invitro data is important, as it provides insights on clinical DDI risk and study design. Historically, the lower limit of plasma fraction unbound (fu,p) is set at 1% for DDI prediction of highly bound compounds by the regulatory agencies due to the uncertainty of the fu,p measurements. This leads to high false positive DDI predictions for highly bound compounds. The recently published ICH M12 DDI guideline allows the use of experimental fu,p for DDI prediction of highly bound compounds. To further build confidence in DDI prediction of highly bound compounds using experimental fu,p values, we evaluated a set of drugs with fu,p < 1% and clinical DDI > 20% using both basic and mechanistic static models. All the compounds evaluated were flagged for DDI risk with the mechanistic model using experimental fu,p values. There was no false negative DDI prediction. Similarly, using the basic model, the DDI risk of all the compounds was identified except for CYP2D6 inhibition of almorexant. The totality of the data demonstrates that the DDI potential of highly bound compounds can be predicted accurately when actual protein binding numbers are measured. [ABSTRACT FROM AUTHOR]

Pre-clinical studies in animals are an essential part of drug development for new chemical entities. Before clinical trials in humans, submission of safety data from one rodent and one non-rodent species is compulsory as per regulatory guidelines. Even though minipigs and monkeys are physiologically closer to humans, dogs are usually employed as the non-rodent pre-clinical species. In this study, the in vitro plasma protein binding of eleven marketed cardiovascular drugs was studied in dog, minipig, monkey and human to determine the preferred species. To conduct plasma protein binding studies, the most reliable equilibrium dialysis method was adopted. Ten out of eleven tested cardiovascular drugs showed statistically similar plasma protein binding in minipig and human plasma which was different from dog and monkey plasma. The results from the studies showed greater similarity between minipigs and humans suggesting that the minipig species maybe a better pre-clinical non-rodent model during drug development of cardiovascular drugs instead of the conventional dog species. Additionally, use of the more accessible minipig species may help in saving time, and resources during pre-clinical studies and may also be more predictive during the safety studies in humans during later stage clinical trials. [ABSTRACT FROM AUTHOR]

BackgroundThe behavior of the comprehensive circulating renin-angiotensin system (RAS) in dogs with myxomatous mitral valve disease (MMVD) before to the onset of congestive heart failure remains largely unexplored.Hypothesis/objectivesThe classical and alternative RAS activity and aldosterone concentrations will be significantly higher in dogs with American College of Veterinary Internal Medicine (ACVIM) stage B2 MMVD compared to normal dogs and dogs with ACVIM stage B1 MMVD.AnimalsOne-hundred seventeen client-owned dogs (normal = 60; B1 = 31; B2 = 26).MethodsProspective observational study. Angiotensin peptides (AP) and aldosterone concentrations were measured using liquid chromatography and mass spectrometry. Angiotensin converting enzymes 1 and 2 (ACE, ACE2) and renin activity surrogates were calculated from AP concentrations. Equilibrium dialysis (ED) and immediate protease inhibition (PI) methods of AP quantification were compared in 14 healthy dogs.ResultsCore RAS activity and aldosterone concentrations did not differ among the 3 groups. However, the balance between the alternative and classical RAS differed, with dogs with stage B2 MMVD having significantly higher ACE2 activity surrogate (ACE2surr ) when compared to normal dogs (adjusted P = .02; ratio of medians for ACE2surr [B2:normal], 1.89; 95% confidence interval [CI]: 1.4-2.6). The ED and PI methods of AP quantification were highly correlated (AngI, r = .9, P

Microsomal binding (MB) is recognized as an important factor in accurately predicting drug-drug interaction. This work investigates differences among various methods employed in MB studies by: • Comparing and contrasts the protocols used and analyzing the data generated. • Identifying potential reasons for the variance in values generated across different methods. • Discussing the clinical and practical implications of selecting the most suitable method for accurate determination of MB. Accurate measurement of non-specific binding of a drug candidate to human liver microsomes (HLM) can be critical for the accurate determination of key enzyme kinetic parameters such as Michaelis-Menton (K m), reversible inhibition (K i), or inactivation (K I) constants. Several methods have been developed to determine non-specific binding of small molecules to HLM, such as rapid equilibrium dialysis (RED), ultrafiltration (UF), HLM bound to magnetizable beads (HLM-beads), ultracentrifugation (UC), the linear extrapolation stability assay (LESA), and the Transil™ system. Despite various differences in methodology between these methods, it is generally presumed that similar free fraction values (f u,mic) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high f u,mic value) and significant (low f u,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The f u,mic values were determined using a single compound concentration (1.0 µM) and three HLM concentrations (0.025, 0.50, and 1.0 mg/mL). When the HLM non-specific binding event is not extensive resulting in high f u,mic values, all methods generated similar f u,mic values. However, f u,mic values varied markedly across assay formats when high binding to HLM occurred, where f u,mic values differed by up to 33-fold depending on the method used. Potential causes for such discrepancies across the various methods employed, practical implications related to conduct the different assays, and implications to clinical drug-drug interaction (DDI) predictions are discussed. [ABSTRACT FROM AUTHOR]

The article focused on a study of the release of melanin from its gel dosage form and the toxicological properties of the gel. The recorded release of melanin from the gel was fastest during the first 3 h (50.8%). The remaining amount of melanin was subsequently released gradually as the system equilibrium shifted. The photoprotective action of the drug was prolonged because of the slowed melanin release. Symptoms of acute toxicosis and lethal outcomes were not recorded in all experimental animals during studies of the acute toxicity parameters of the developed gel based on melanin isolated from horse chestnut seed episperm with subcutaneous administration and cutaneous application at doses of 1500, 2000, 2500, and 3000 mg/kg. The developed photoprotective gel belonged to class Vof practically harmless substances. The LD50 value for cutaneous application to rats was >2500 mg/kg. [ABSTRACT FROM AUTHOR]

Developing procedures based on equilibrium dialysis (ED) that allow measuring the free drug concentration in plasma improves therapeutic drug monitoring (TDM) in those cases where its measurement is justified. However, this procedure requires specific sample preparation and presents different pitfalls, which are not error-free. As with any result provided by a clinical laboratory, this one should be as accurate as possible to allow a correct clinical interpretation. The measurement uncertainty (MU) is a parameter that enables the accuracy of results to be known, and that is mandated by ISO 15189. Herein, this study suggests how the MU for the results of the free drug concentrations in serum could be estimated when an ED is used. A combination of the top-down and bottom-up approaches was used to estimate the MU based on the ISO/TS 20914:2019 and JCGM 100:2008 guidelines, including the concentration of free phenytoin in serum, as an example. Different scenarios were incorporated considering or not a significant bias related to the primary drawbacks of ED: the non-specific binding, the volume shift effect and the Gibbs-Donnan effect. The expanded uncertainties estimated ranged between 13.0 and 30.9 %. The highest MU corresponded to the free drug concentrations in serum results when significant biases related to the volume shift and Gibbs-Donnan effects exist. A detailed estimation of MU for free drug concentrations is presented using ED, considering different scenarios. This study could stimulate clinical laboratories to perform MU studies and its application in TDM. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of fluorinated compounds which have yet to undergo comprehensive investigation regarding potential adverse health effects and bioaccumulative properties. With long half-lives and accumulative properties, PFAS have been linked to several toxic effects in both non-clinical species such as rat and mouse as well as human. Although biological impacts and specific protein binding of PFAS have been examined, there is no study focusing on the species-specific fraction unbound (fu) in plasma and related toxicokinetics. Herein, a presaturation equilibrium dialysis method was used to measure and validate the binding of 14 individual PFAS with carbon chains containing 4 to 12 perfluorinated carbon atoms and several functional head-groups to albumin and plasma of mouse (C57BL/6 and CD-1), rat, and human. Equivalence testing between each species-matrix combination showed positive correlation between rat and human when comparing fu in plasma and binding to albumin. Similar trends in binding were also observed for mouse plasma and albumin. Relatively high Spearman correlations for all combinations indicate high concordance of PFAS binding regardless of matrix. Physiochemical properties of PFAS such as molecular weight, chain length, and lipophilicity were found to have important roles in plasma protein binding of PFAS. [ABSTRACT FROM AUTHOR]

Moxidectin (MOX) is a macrocyclic lactone used to eliminate endo and ectoparasites in many mammalian species. It is notably the active ingredient of the anti-parasitic drug Cydectin®, manufactured by Virbac, and is frequently used to treat sarcoptic mange in Australian wildlife. Protein binding plays a significant role in the efficacy of a drug, as the unbound/free drug in plasma ultimately reflects the pharmacologically relevant concentration. This study aimed to investigate the free drug percentage of Moxidectin after in vitro spiking into the sera of four sarcoptic mange-susceptible Australian wildlife species; the koala (Phascolarctos cinereus), the bare-nosed wombat (Vombatus ursinus), the eastern grey kangaroo (Macropus giganteus), and the mountain brushtail possum (Trichosurus cunninghami). Three concentration points of MOX were tested for each individual: 20 pg/μL, 100 pg/μL and 500 pg/μL. Serum from five individuals of each species underwent an equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed an atypical concentration dependent binding across all species, where free drug percentage decreased as MOX concentration increased. In addition, wombats showed significantly lower free drug levels. These findings call for further research into the mechanisms of moxidectin protein binding to help understand MOX pharmacokinetics in marsupials.

Background: Free thyroxine (FT4) measurement is one of the most requested tests in patient care for diagnosing and treating thyroid-related illnesses. Equilibrium dialysis (ED) is considered the “gold standard” for FT4 measurement; however, several factors have a profound effect on the reliability of FT4 assays and require special consideration. Methods: In the current study, we focused on evaluating critical factors that could contribute to reporting errors, such as adsorption of thyroxine (T4) to labware surfaces, stability of serum samples, stock solutions, and calibrator storage conditions, as well as the solvents used to prepare T4 solutions. Results: The adsorption of T4 in ethanolic solutions and dialysates to labware surfaces can be reduced with the careful selection of pipette tips, test tubes, and 96-well plates. Adding pH modifiers to neat T4 solutions can improve its stability. FT4 in serum samples remains stable after exposure to four freeze–thaw cycles, 5 °C for 18–20 h, or −70 °C for a minimum of three years. Conclusion: The presented study has demonstrated that the loss of analyte due to pre-analytical and analytical factors during operation of the FT4 reference measurement procedure (RMP) can be minimized by careful selection of all labware for sample preparation. It was found that the accuracy and imprecision of FT4 assays can be influenced by different types of dialysis devices, but acceptable alternatives to ED membranes were identified. This study demonstrates approaches to establish a FT4 method that is independent from specific suppliers and addresses critical pre-analytical and analytical factors important for FT4 measurements.

Drinking water contaminated by fluorosurfactant-based aqueous film-forming foams (AFFF) is a source of human exposure to poly- and perfluoroalkyl substances (PFAS). However, assessment of bioaccumulation potentials of diverse PFAS in commercial products such as AFFF have been insufficient and challenging, especially due to a lack of analytical standards. Here we explore the value of suspect screening, equilibrium dialysis, and molecular-docking simulations to identify potentially bioaccumulative PFAS. We exposed human serum albumin (HSA) protein to dilutions of a legacy AFFF produced by 3M in 1999 using equilibrium dialysis and screened in-vitro protein-binding affinities using high-resolution mass spectrometry (HRMS). Through suspect screening, we identified 32 PFAS and 18 hydrocarbon surfactants in the AFFF that bound to HSA. Quantification of noncovalent association constants for 26 PFAS standards confirmed that many PFAS, including the short-chain perfluoropropane sulfonic acid (log Ka= 4.1 ± 0.2 M-1), exhibit strong binding affinities with HSA. At least five PFAS in AFFF (including three PFAS with less than five perfluorocarbons) remained bound to the precipitated HSA pellet after extensive solvent washing-an indication of high PFAS binding potential. Three PFAS (PFBS, PFOS, and PFOA) were confirmed in the protein pellet with analytical standards and quantified after acid digestion-this sample fraction accounted for 5 to 20% of each compound mass in the sample. We calculated pseudo-bioconcentration factors (BCFpseudo) for PFAS that suspect screening flagged as noncovalently bound or potentially covalently bound. Most PFAS exhibiting high BCFpseudo, especially those with seven perfluorocarbons, contained a carboxylic acid or a sulfonic acid. Finally, we used molecular docking to simulate HSA binding affinities for 62 ligands (26 PFAS targets, 18 PFAS qualified in AFFF, and 18 hydrocarbon surfactants qualified in AFFF). We found that molecular docking can effectively separate HSA-binding and -nonbinding compounds in AFFF. In-vitro and in-silico approaches described in this study provide replicable, high-throughput workflows for assessing bioaccumulation potentials of diverse PFAS in commercial products.

• The addition of an organophosphate esterase inhibitor, dichlorvos, stabilized remdesivir (RDV) during equilibrium dialysis for plasma protein binding (PPB) determination. • Dichlorvos did not impact the unbound fraction (f u) of RDV in plasma, and an equilibrium dialysis method for RDV was developed with the addition of dichlorvos. • Tenofovir alafenamide (TAF) was more stable than RDV by a factor of 3 but was not stabilized by dichlorvos. Fit-for-purpose acceptance criteria for equilibrium dialysis (ED) were implemented during PPB method development for TAF. • PPB methods for RDV and TAF were qualified and applied to PPB determinations for clinical studies. Remdesivir (RDV) and tenofovir alafenamide (TAF) are prodrugs designed to be converted to their respective active metabolites. Plasma protein binding (PPB) determination of these prodrugs is important for patients with possible alteration of free fraction of the drugs due to plasma protein changes in renal impairment, hepatic impairment, or pregnancy. However, the prodrugs' instability in human plasma presents a challenge for accurate PPB determination. In this research work, two approaches were used in the method development and qualification for PPB assessment of RDV and TAF. For RDV, dichlorvos was used to inhibit esterase activity to stabilize the prodrug in plasma during equilibrium dialysis (ED). The impact of dichlorvos on protein binding was evaluated and determined to be insignificant by comparing the unbound fraction (f u) determined by the ED method with dichlorvos present and the f u determined by an ultrafiltration method without dichlorvos. In contrast to RDV, TAF degradation in plasma is ∼3-fold slower, and TAF stability cannot be improved by dichlorvos. Fit-for-purpose acceptance criteria for the TAF PPB method were chosen, and an ED method was developed based on these criteria. These two methods were then qualified and applied for PPB determinations in clinical studies. [ABSTRACT FROM AUTHOR]

Background: Changes in cortisol binding globulin (CBG) impact the total serum cortisol concentration and affect the accurate assessment of adrenal function. Free biologically cortisol can be calculated using different equations or directly measured after complicated procedures. Methods: The free cortisol index (FCI) obtained using the Bonte formula as well as the free cortisol concentration calculated (Coolens equation) were first estimated for 45 healthy workers. The CBG level was determined by a competitive radioimmunoassay and the total cortisol concentration, was measured with an electrochemiluminescent assay. The correlations between FCI, the free cortisol concentrations calculated and the free cortisol levels measured with liquid chromatography-tandem mass spectrometry after equilibrium dialysis were studied for those 45 samples. Reference limits were established on 158 healthy hospital workers and patients with serum samples collected between 7:30 am and 10 am. Results: The FCI as well as the free cortisol concentrations calculated obtained for the 45 samples correlated significantly with the free cortisol levels measured. Although the cortisol and CBG levels were statistically higher in women using contraceptives compared with women not taking them as well as men, the calculated FCI and free cortisol concentrations did not differ between these groups. The medians (P2.5-P97.5) obtained for the 158 healthy workers were respectively 26.4% (12.3-51.6%) and 10.6 nmol/L (4.3-26.7 nmol/L). Conclusions: This study highlighted a significant correlation between the FCI, the free cortisol concentrations calculated and the free cortisol levels measured with LC-MS/MS, it has also allowed the establishment of reference intervals for calculated FCI and free cortisol. [ABSTRACT FROM AUTHOR]

Equilibrium dialysis (ED) is widely used in pharmacokinetics to determine the fraction of unbound (f u) compounds in plasma; however, the kinetics of drugs in the ED system with respect to their permeation across semi-permeable membranes has not been systemically studied. Here, the kinetics of the ED system, including the binding of drugs to plasma proteins, non-specific binding, and permeation across the membrane, was described to enable verification of the equilibrium, prediction of the time to reach equilibrium, and estimations of f u with data obtained during pre-equilibrium. Using data obtained during pre-equilibrium, the time to reach 90% equilibrium (t 90 %) and f u were estimated with reasonable accuracy. Notably, f u could be estimated reasonably well using one-time-point data for the calculation. Furthermore, the current modeling approach allowed concurrent estimations of f u and the decomposition rate of compounds that were metabolically unstable in the plasma. Reasonable metabolic rate constants were determined for cefadroxil and diltiazem, demonstrating the practicality of this method for determining kinetics related to f u characterization. Because the determination of f u of compounds with 'unfavorable' physicochemical properties is known to be experimentally challenging, the current method may be useful in determining the f u of compounds in vitro. [ABSTRACT FROM AUTHOR]

Background:The selective occurrence of hepatotoxicity observed with use of pazopanib may be attributed to its high level of plasma protein binding and low hepatic extraction ratio. The primary objective was to investigate changes in free drug concentration amongst patients with varying albumin concentrations. Methods:A HPLC-MS/MS method using C18 column (4.6 × 150 mm, 5 μm) with ESI source in positive mode had been developed and validated for the quantitative determination of free pazaopanib concentration in human plasma. Prior to sample preparation, patient samples were subjected to 6-hour equilibrium dialysis with molecular weight cut-off set at 8000 Da. Results:The calibration curves were linear over the range of 5-1000 ng/mL, with a lower limit of quantification of 5 ng/mL. The intra-day and inter-day precisions and accuracies were all within ± 15 %, at 3 different quality controls. Higher median fraction unbound of pazopanib were observed in patients (n = 17) with lower than normal albumin concentrations. Conclusion:With the developed assay, monitoring of plasma free concentrations may be evaluated as an indicator of pazopanib exposure in patients.

Abstract Background The behavior of the comprehensive circulating renin‐angiotensin system (RAS) in dogs with myxomatous mitral valve disease (MMVD) before to the onset of congestive heart failure remains largely unexplored. Hypothesis/Objectives The classical and alternative RAS activity and aldosterone concentrations will be significantly higher in dogs with American College of Veterinary Internal Medicine (ACVIM) stage B2 MMVD compared to normal dogs and dogs with ACVIM stage B1 MMVD. Animals One‐hundred seventeen client‐owned dogs (normal = 60; B1 = 31; B2 = 26). Methods Prospective observational study. Angiotensin peptides (AP) and aldosterone concentrations were measured using liquid chromatography and mass spectrometry. Angiotensin converting enzymes 1 and 2 (ACE, ACE2) and renin activity surrogates were calculated from AP concentrations. Equilibrium dialysis (ED) and immediate protease inhibition (PI) methods of AP quantification were compared in 14 healthy dogs. Results Core RAS activity and aldosterone concentrations did not differ among the 3 groups. However, the balance between the alternative and classical RAS differed, with dogs with stage B2 MMVD having significantly higher ACE2 activity surrogate (ACE2surr) when compared to normal dogs (adjusted P = .02; ratio of medians for ACE2surr [B2:normal], 1.89; 95% confidence interval [CI]: 1.4‐2.6). The ED and PI methods of AP quantification were highly correlated (AngI, r = .9, P

The confidence in fraction unbound (ƒ u) using equilibrium dialysis (ED) is often questioned (e.g., highly bound, labile compounds) due to uncertainty in whether true equilibrium is achieved. Different methods have been developed to increase confidence in ƒ u measurements, such as the presaturation, dilution, and bi-directional ED methods. However, confidence in ƒ u measurement can still suffer due to non-specific binding and inter-run variations introduced during equilibrium and analysis. To address this concern, we introduce an orthogonal approach called counter equilibrium dialysis (CED) in which non-labeled and isotope-labeled compounds are dosed counter-directionally in rapid equilibrium dialysis (RED). ƒ u values of both non-labeled and labeled compounds are measured simultaneously in the same run. These tactics not only minimize non-specific binding and inter-run variability but also enable the confirmation of true equilibrium. If equilibrium is reached in both dialysis directions, the ƒ u for the non-labeled compound and the labeled compound will converge. The refined methodology was extensively tested with various compounds of diverse physicochemical properties and plasma binding characteristics. Our results demonstrated that, by using the CED method, ƒ u values for a wide range of compounds could be accurately determined with significantly improved confidence, including the challenging highly bound and labile compounds. [ABSTRACT FROM AUTHOR]

• A highly accurate and precise candidate Reference Measurement Procedure (cRMP) was developed for CDC Clinical Standardization Programs (CDC CSP) free thyroxine (FT4) measurements. • The FT4 cRMP was based on equilibrium dialysis (ED) and isotope dilution liquid chromatography – tandem mass spectrometry (ID-LC-MS/MS) procedures. • The accuracy and precision of the cRMP were validated via an interlaboratory comparison study with excellent agreement between four independent laboratories. • The low limit of detection of cRMP provided sufficient sensitivity to determine FT4 for patients with hypothyroidism. Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays. [ABSTRACT FROM AUTHOR]

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of fluorinated compounds which have yet to undergo comprehensive investigation regarding potential adverse health effects and bioaccumulative properties. With long half-lives and accumulative properties, PFAS have been linked to several toxic effects in both non-clinical species such as rat and mouse as well as human. Although biological impacts and specific protein binding of PFAS have been examined, there is no study focusing on the species-specific fraction unbound (fu) in plasma and related toxicokinetics. Herein, a presaturation equilibrium dialysis method was used to measure and validate the binding of 14 individual PFAS with carbon chains containing 4 to 12 perfluorinated carbon atoms and several functional head-groups to albumin and plasma of mouse (C57BL/6 and CD-1), rat, and human. Equivalence testing between each species-matrix combination showed positive correlation between rat and human when comparing fu in plasma and binding to albumin. Similar trends in binding were also observed for mouse plasma and albumin. Relatively high Spearman correlations for all combinations indicate high concordance of PFAS binding regardless of matrix. Physiochemical properties of PFAS such as molecular weight, chain length, and lipophilicity were found to have important roles in plasma protein binding of PFAS.

Background: Free testosterone (FT) determination may be helpful in evaluating men suspected of testosterone deficiency especially in conditions with altered binding‐protein concentrations. However, methods for measuring FT by equilibrium dialysis and reference intervals vary among laboratories. Objective: To determine reference intervals for FT in healthy, nonobese men by age groups as well as in healthy young men, 19–39 years, using a standardized equilibrium dialysis procedure Methods: We measured FT in 145 healthy, nonobese men, 19 years or older, using a standardized equilibrium dialysis method performed for 16‐h at 37°C using undiluted serum and dialysis buffer that mimicked the ionic composition of human plasma. FT in dialysate was measured using a CDC‐certified liquid chromatography tandem mass spectrometry assay. Results: In healthy nonobese men, the 2.5th, 10th, 50th, 90th, and 97.5th percentile values for FT were 66, 91, 141, 240, and 309 pg/ml, respectively; corresponding values for men, 19–39 years, were 120, 128, 190, 274, and 368 pg/ml, respectively. FT levels by age groups exhibit the expected age‐related decline. FT levels were negatively associated with body mass index, age, and sex hormone‐binding globulin (SHBG) levels. Percent FT was lower in middle‐aged and older men than young men adjusting for SHBG level. Discussion: Further studies are needed to determine how these reference intervals apply to the diagnosis of androgen deficiency in clinical populations and in men of different races and ethnicities in different geographic regions. Conclusion: Reference intervals for free FT levels (normative range 66–309 pg/ml [229–1072 pmol/L] in all men and 120–368 pg/ml [415–1274 pmol/L] in men, 19–39 years), measured using a standardized equilibrium dialysis method in healthy nonobese men, provide a rational basis for categorizing FT levels. These intervals require further validation in other populations, in relation to outcomes, and in randomized trials. [ABSTRACT FROM AUTHOR]

Objective: To characterize the circulating androgen levels across the menstrual cycle in healthy women using highly sensitive and accurate methods and report sex differences in the relative levels of dihydrotestosterone (DHT) to testosterone (T) levels.Design: Prospective cohort study.Setting: Research clinic, academic teaching hospital.Patient(s): Twenty-one healthy premenopausal women, aged 19-40 years, with regular menstrual cycles.Intervention(s): Not applicable.Main Outcome Measure(s): Serum total T and DHT levels measured using liquid chromatography-tandem mass spectrometry, free T levels measured using a standardized equilibrium dialysis method coupled with measurement of the T levels in the dialysate using liquid chromatography-tandem mass spectrometry, and comparison of the DHT-to-T ratio between healthy women and age-matched healthy men.Result(s): The serum total and free T levels increased across the follicular phase and peaked at midcycle (total T, 43.6 ± 16.2 ng/dL; free T, 15.6 ± 11.9 pg/mL) and gradually declined in the luteal phase. The DHT level did not significantly change across the menstrual cycle. The DHT-to-T ratios were 1:4 and 1:13 in women and men, respectively.Conclusion(s): In healthy premenopausal women, the total and free T levels varied significantly across the menstrual cycle, whereas the DHT levels did not change; the peak total and free T levels in the midcycle period were higher than previously reported, underscoring the importance of establishing menstrual phase-specific reference ranges to avoid misdiagnosis of hyperandrogenism. Women have significantly higher DHT levels relative to total T than men; the significance of this sex difference in the DHT-to-T ratio needs further investigation. [ABSTRACT FROM AUTHOR]

LS-102 is a new derivative of astragaloside IV (AGS IV) that has been shown to possess potentially significant cardioprotective effects. However, there are no reports concerning its interaction with human serum albumin (HSA) and toxicology in vertebrates. The present investigation was undertaken to characterize the interaction of AGS IV and LS-102 with HSA using equilibrium dialysis and UHPLC-MS/MS methods, along with computational methods. Notably, the effects of AGS IV and LS-102 were studied in vivo using the zebrafish embryo model. Markers related to embryonic cardiotoxicity and thrombosis were evaluated. We showed that the plasma protein binding rate of AGS IV (94.04%-97.42%) was significantly higher than that of LS-102 (66.90%-69.35%). Through site marker competitive experiments and molecular docking, we found that AGS IV and LS-102 were located at the interface of subdomains IIA and IIIA, but the site I might be the primary binding site. Molecular dynamics revealed that AGS IV showed a higher binding free energy mainly due to the stronger hydrophobic and hydrogen bonding interactions. Moreover, the secondary structure implied no obvious effect on the protein structure and conformation during the binding of LS-102. LS-102 significantly ameliorated the astramizole-induced heart rate slowing, increased SV-BA spacing, and prevented arachidonic acid-induced thrombosis in zebrafish. To our knowledge, we are the first to reveal that LS-102 binds to HSA with reversible and moderate affinity, indicating its easy diffusion from the circulatory system to the target tissue, thereby providing significant insights into its pharmacokinetic and pharmacodynamic properties when spread in the human body. Our results also provide a reference for the rational clinical application of LS-102 in the cardiovascular field. [ABSTRACT FROM AUTHOR]

A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of total and unbound concentrations of LY3214996, an extracellular signal-regulated kinase inhibitor; abemaciclib, a cyclin-dependent kinase 4/6 inhibitor; and abemaciclib active metabolites, M2 and M20, in human plasma, brain tumor, and cerebrospinal fluid samples. The method was validated over a concentration range of 0.2–500 nM within a total run time of 3.8 min using isocratic elution on a Kinetex™ F 5 column. Detection was performed on a Sciex QTRAP 6500+ mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. The intra- and inter-batch accuracy as well as the precision of the method for all matrices was within ±20% and ≤20% at the lower limit of quantification, and within ±15% and ≤15% for other quality control levels for all analytes. The unbound fractions of drugs and metabolites in spiked and patient samples were determined using an optimized equilibrium dialysis. The validated method was successfully applied in a phase 0/2 clinical trial to assess the central nervous system penetration of LY3214996 and abemaciclib. [Display omitted] • The LC-MS/MS method was validated to measure LY3214996, abemaciclib, M2 and M20 in human plasma, brain tumor, and CSF. • Unbound fractions of LY3214996, abemaciclib, M2 and M20 were established in human plasma, brain, and brain tumor. • The total and unbound levels of LY3214996, abemaciclib, M2 and M20 in glioblastoma patients have been reported. [ABSTRACT FROM AUTHOR]

Background/Introduction: Plasma protein binding (PPB) continues to be a key aspect of antibiotic development and clinical use. PPB is essential to understand several properties of drug candidates, including antimicrobial activity, drug-drug interaction, drug clearance, volume of distribution, and therapeutic index. Focus areas of the review: In this review, we discuss the basics of PPB, including the main drug binding proteins i.e., Albumin and α-1-acid glycoprotein (AAG). Furthermore, we present the effects of PPB on the antimicrobial activity of antibiotics and the current role of PPB in in vitro pharmacodynamic (PD) models of antibiotics. Moreover, the effect of PPB on the PK/PD of antibiotics has been discussed in this review. A key aspect of this paper is a concise evaluation of PPB between animal species (dog, rat, mouse, rabbit and monkey) and humans. Our statistical analysis of the data available in the literature suggests a significant difference between antibiotic binding in humans and that of dogs or mice, with the majority of measurements from the pre-clinical species falling within five-fold of the human plasma value. Conversely, no significant difference in binding was found between humans and rats, rabbits, or monkeys. This information may be helpful for drug researchers to select the most relevant animal species in which the metabolism of a compound can be studied for extrapolating the results to humans. Furthermore, state-of-the-art methods for determining PPB such as equilibrium dialysis, ultracentrifugation, microdialysis, gel filtration, chromatographic methods and fluorescence spectroscopy are highlighted with their advantages and disadvantages. [ABSTRACT FROM AUTHOR]

The present study showed the binding interaction of 2-(4,5-Dihydro-1,2-oxazol-5-yl)-phenol-N-methylaniline (2DHOPNA) with BSA in 1,4-dioxane, DMSO and DMF by equilibrium dialysis, FT-IR, acoustical at physiological pH and its molecular modeling study. Findings were interpreted by scatchard plot which showed an increase in association constants with increasing temperature and concentrations of the 2DHOPNA. It is seen that, the binding supposed to be more significant in 1,4-dioxane than DMSO and DMF. Thermodynamic parameters are also determined for the binding interaction of 2DHOPNA with BSA. Values of Gibb’s free energy (∆G), enthalpy (∆H) and entropy (∆S) were calculated by using van’t Hoff equation. The positive values of ∆H and ∆S showed exothermic interaction between 2DHOPNA and BSA. Similarly, negative ∆G showed the spontaneity of the binding process. ∆G becomes more negative with increase in temperature, indicated feasibility of the binding interaction at high temperature. Molecular modeling confirmed the binding interaction having energy of -210.13. DOI: http://dx.doi.org/10.17807/orbital.v12i3.1450

Platinum (Pt) antineoplastic agents remain indispensable for the treatment of oncological disease. Pt-based drugs are mainly used in the therapy of ovarian cancer and non-small-cell lung carcinoma. A novel platinum-containing antineoplastic agent BP-C1 is a complex of diamminoplatinum with an oxygen-donor polymeric ligand of benzene-polycarboxylic acids, isolated from natural lignin. The aim of the study was to investigate ex vivo protein binding of BP-C1. Protein binding of BP-C1 was tested using equilibrium dialysis. Pooled blood plasma was used in the study. Control solutions contained the same dosages of BP-C1 in PBS (pH 7.2). Plasma and control solutions were submitted to equilibrium dialysis across a vertical 8 kDa cut-off membrane for 4 h at 37 °C under gentle shaking. Platinum was quantified in dialysis and retained fractions using inductively coupled plasma mass spectrometry after microwave digestion. The dialysis system was tested and validated; this showed no protein saturation with platinum. A medium degree of binding of platinum to macromolecular species of ca. 60% was observed. The study showed the maintenance of a high fraction of free BP-C1 in the bloodstream, facilitating its pharmacological activity. [ABSTRACT FROM AUTHOR]

[Display omitted] • fluorescence intensity ratio decreases with increasing SRH for HSA. • HSA fluorescence was quenched by SRH via a dynamic quenching mechanism. • The Scatchard plot and Hill coefficient obtain by Equilibrium dialysis. • Increasing the molar ratio of SRH: HSA increased the α-helix by FT-IR and CD. In this study, the interaction between sertraline hydrochloride (SRH), an antidepressant drug, and human serum albumin (HSA) in a physiological buffer was investigated using UV–Vis spectroscopy, equilibrium dialysis, fluorescence emission spectroscopy, circular dichroism, Fourier transform infrared (FT-IR) spectroscopy, and molecular docking methods. The results showed that SRH induced fluorescence quenching of HSA through a mixed mechanism. The drug-protein binding process was a cooperative binding pattern, as confirmed by the Scatchard plot and Hill coefficient. Molecular docking results demonstrated that SRH preferentially binds to site I in subdomain IIA of HSA, where Trp-214 is located, as confirmed by fluorescence analysis. Additionally, the binding constant (K b), number of binding sites (n), and thermodynamic parameters showed that the binding of SRH to HSA was spontaneous, and that hydrophobic interactions were the main forces of this interaction. Furthermore, the results showed a conformational change in HSA upon interaction with SRH, as revealed by UV–Vis, circular dichroism, and Fourier transform infrared spectroscopy. [ABSTRACT FROM AUTHOR]

Niraparib is a potent and orally bioavailable inhibitor of poly (ADP-ribose) polymerase (PARP) with high specificity for isoforms 1 and 2. It has been approved by the U.S. Food and Drug Administration for ovarian cancer maintenance therapy and is currently under development for various cancers, including glioblastoma. To assess central nervous system (CNS) penetration of niraparib in glioblastoma patients, a novel bioanalytical method was developed to measure total and unbound niraparib levels in human brain tumor tissue and cerebrospinal fluid (CSF). The method was validated using plasma as a surrogate matrix over the concentration range of 1–10,000 nM on an LC-MS/MS system. The MS/MS detection was conducted in positive electrospray ionization mode, while chromatography was performed using a Kinetex™ PS C18 column with a total 3.5-minute gradient elution run time. The maximum coefficient of variation for both intra- and inter-day precision was 10.6%, with accuracy ranging from 92.8% – 118.5% across all matrices. Niraparib was stable in human brain homogenate for at least 6 hours at room temperature (RT) and 32 days at −20°C, as well as in stock and working solutions for at least 21 hours (RT) and 278 days (4°C). Equilibrium dialysis experiments revealed the fractions unbound of 0.05 and 0.16 for niraparib in human brain and plasma, respectively. The validated method is currently employed to assess niraparib levels in human glioblastoma tissue, CSF, and plasma in an ongoing trial on newly diagnosed glioblastoma and recurrent IDH1/2(+) ATRX mutant glioma patients (NCT05076513). Initial results of calculated total (K p) and unbound (K p,uu) tumor-to-plasma partition coefficients indicate significant brain penetration ability of niraparib in glioblastoma patients. [Display omitted] • The LC-MS/MS method was validated to measure niraparib levels in human plasma, brain tumor, and CSF. • Unbound fractions of niraparib were established in human plasma, brain, and brain tumor. • The total and unbound levels of niraparib in glioblastoma patients have been evaluated. [ABSTRACT FROM AUTHOR]

Background contextIt is well-established that disc mechanical properties degrade with degeneration. However, prior studies utilized cadaveric tissues from donors with undefined back pain history. Disc degeneration may present with pain at the affected motion segment, or it may be present in the absence of back pain. The mechanical properties and matrix quantity of discs removed and diagnosed for degeneration with patient chronic pain may be distinct from those with other diagnoses, such as spinal deformity.PurposeTo test the hypothesis that discs from nondeformity segments have inferior mechanical properties than deformity discs owing to differences in matrix quality.Study design/settingIn vitro study comparing the mechanical and matrix properties of discs from surgery patients with spinal nondeformity and deformity.MethodsWe analyzed nucleus and annulus samples (8-11 specimens per group) from surgical discectomy patients as part of a fusion or disc replacement procedure. Tissues were divided into two cohorts: nondeformity and deformity. Dynamic indentation tests were used to determine energy dissipation, indentation modulus, and viscoelasticity. Tissue hydration at a physiologic pressure was assessed by equilibrium dialysis. Proteoglycan, collagen, and collagen cross-link content were quantified. Matrix structure was assessed by histology.ResultsWe observed that energy dissipation was significantly higher in the nondeformity nucleus than in the deformity nucleus. Equilibrium dialysis experiments showed that annulus swelling was significantly lower in the nondeformity group. Consistent with this, we observed that the nondeformity annulus had lower proteoglycan and higher collagen contents.ConclusionsOur data suggest that discs from nondeformity discs have subtle differences in mechanical properties compared with deformity discs. These differences were partially explained by matrix biochemical composition for the annulus, but not for the nucleus. The results of this study suggest that compromised matrix quality and diminished mechanical properties are features that potentially accompany discs of patients undergoing segmental fusion or disc replacement for disc degeneration and chronic back pain. These features have previously been implicated in pain via instability or reduced motion segment stiffness.

Background/Introduction: Plasma protein binding (PPB) continues to be a key aspect of antibiotic development and clinical use. PPB is essential to understand several properties of drug candidates, including antimicrobial activity, drug-drug interaction, drug clearance, volume of distribution, and therapeutic index. Focus areas of the review: In this review, we discuss the basics of PPB, including the main drug binding proteins i.e., Albumin and α-1-acid glycoprotein (AAG). Furthermore, we present the effects of PPB on the antimicrobial activity of antibiotics and the current role of PPB in in vitro pharmacodynamic (PD) models of antibiotics. Moreover, the effect of PPB on the PK/PD of antibiotics has been discussed in this review. A key aspect of this paper is a concise evaluation of PPB between animal species (dog, rat, mouse, rabbit and monkey) and humans. Our statistical analysis of the data available in the literature suggests a significant difference between antibiotic binding in humans and that of dogs or mice, with the majority of measurements from the pre-clinical species falling within five-fold of the human plasma value. Conversely, no significant difference in binding was found between humans and rats, rabbits, or monkeys. This information may be helpful for drug researchers to select the most relevant animal species in which the metabolism of a compound can be studied for extrapolating the results to humans. Furthermore, state-of-the-art methods for determining PPB such as equilibrium dialysis, ultracentrifugation, microdialysis, gel filtration, chromatographic methods and fluorescence spectroscopy are highlighted with their advantages and disadvantages.

Adverse health effects associated with exposures to perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a concern for public health and are driven by their elimination half-lives and accumulation in specific tissues. However, data on PFAS binding in human tissues are limited. Accumulation of PFAS in human tissues has been linked to interactions with specific proteins and lipids in target organs. Additional data on PFAS binding and unbound fractions ( f ) in whole human tissues are urgently needed. Here, we address this gap by using rapid equilibrium dialysis to measure the binding and unbound ) in whole human tissues are urgently needed. Here, we address this gap by using rapid equilibrium dialysis to measure the binding and f unbound of 16 PFAS with 3 to 13 perfluorinated carbon atoms (η pfc = 3-13) and several functional headgroups in human liver, lung, kidney, heart, and brain tissue. We compare results to mouse (C57BL/6 and CD-1) and rat tissues. Results show that f unbound decreases with increasing fluorinated carbon chain length and hydrophobicity. Among human tissues, PFAS binding was generally greatest in brain > liver ≈ kidneys ≈ heart > lungs. A correlation analysis among human and rodent tissues identified rat liver as a suitable surrogate for predicting f unbound for PFAS in human tissues ( R 2 ≥ 0.98). The f unbound data resulting from this work and the rat liver prediction method offer input parameters and tools for toxicokinetic models for legacy and emerging PFAS.

Background: The selective occurrence of hepatotoxicity observed with use of pazopanib may be attributed to its high level of plasma protein binding and low hepatic extraction ratio. The primary objective was to investigate changes in free drug concentration amongst patients with varying albumin concentrations. Methods: A HPLC-MS/MS method using C18 column (4.6 × 150 mm, 5 μm) with ESI source in positive mode had been developed and validated for the quantitative determination of free pazaopanib concentration in human plasma. Prior to sample preparation, patient samples were subjected to 6-hour equilibrium dialysis with molecular weight cut-off set at 8000 Da. Results: The calibration curves were linear over the range of 5–1000 ng/mL, with a lower limit of quantification of 5 ng/mL. The intra-day and inter-day precisions and accuracies were all within ± 15 %, at 3 different quality controls. Higher median fraction unbound of pazopanib were observed in patients (n = 17) with lower than normal albumin concentrations. Conclusion: With the developed assay, monitoring of plasma free concentrations may be evaluated as an indicator of pazopanib exposure in patients.

This paper presented the comparative binding interaction of ethyl-4-(4-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4-HP2OTP) and ethyl-4-(2-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2-HP2STP) to bovine serum albumin (BSA) in 1,4-dioxane, DMSO and DMF by equilibrium dialysis, FT-IR and acoustical study at physiological pH. The binding data obtained was interpreted by scatchard plot, which gives the association constants. An increase in association constants is observed with increase in temperature and concentration. FT-IR study explains the binding through shifting in peak positions of amide I and II. It explained the changes in secondary structure of BSA on binding with the drugs. The free energy (∆G), enthalpy (∆H) and entropy (∆S) values were calculated by using van’t Hoff equation. The negative ∆G showed the spontaneous process and positive values of ∆H and ∆S showed endothermic interaction between ligands and BSA. ∆G becomes more negative with increased in temperature, indicated feasibility of binding interaction at high temperature. The positive values of ∆H and ∆S also showed specific electrostatic and hydrophobic interaction between ligand and BSA. DOI: http://dx.doi.org/10.17807/orbital.v10i2.1116

Objectives: The first objective was to assess correlation between free thyroxine (fT4) measurements by equilibrium dialysis (fT4ED; Antech Diagnostics) and a chemiluminescent enzyme immunoassay (fT4CEIA; IMMULITE 2000 Veterinary Free T4 [Siemens Healthcare Diagnostics Products]) in hyperthyroid, otherwise healthy, cats before (T0), and 1 month (T1) and 11–23 months (T2) after radioactive iodine (131I) therapy. The second objective was to determine correlation between thyroid status based on fT4 (by both techniques) and the gold standard, thyroid scintigraphy. Methods: Thyroid status, including thyroid-stimulating hormone (TSH), total thyroxine (TT4) and fT4 serum concentrations, were assessed in 45 client-owned hyperthyroid cats before (T0), and 1 month (T1) and 11–23 months (T2) after 131I therapy. fT4 was determined by a chemiluminescent enzyme immunoassay (CEIA) and equilibrium dialysis (ED). Quantitative thyroid scintigraphy (with sodium 99m-Tc-pertechnetate) was performed at T2. Results: Spearman correlation between fT4CEIA and fT4ED was 0.81, 0.88 and 0.79 at T0, T1 and T2, respectively. fT4CEIA was consistently lower than fT4ED, with a median difference of −5.4 pmol/l (P <0.001) and −4.9 pmol/l (P <0.0001) at T1 and T2, respectively. At T2, all cats were identified as euthyroid based on thyroid scintigraphy. None of the cats were identified as being hypothyroid, based on serum TT4 and TSH measurements. Nine of 22 (40.9%) cats had an fT4CEIA below the reference interval (RI) at T2, whereas only 2/22 (9.1%) cats had an fT4ED concentration below the RI at T2. Conclusions and relevance: Good correlation exists between both assays at T1 and T2, but a significant systematic difference is noted at both time points. This could be an indication for reconsideration of the current RI, although further studies are warranted for assessing test accuracy (in otherwise healthy cats and cats with non-thyroidal illness). At this time, routine use of fT4CEIA after 131I therapy is not advised in feline patients. [ABSTRACT FROM AUTHOR]

The use of IBH-5 decreased the kdeg values and increased the half-life of the compounds PNZ, TCP, Cpd I and Cpd II with kdeg values of 1.10 × 10−4 s− 1 (t1/2= 115 min), 4 × 10−5 s−1 (t1/2 = 289 min), 4 × 10−5 s−1 (t1/2 = 289 min), and 3 × 10−5 s−1 (t1/2 = 385 min) respectively, compared to kdeg values of 1.25 × 10−2 s−1 (t1/2 = 0.9 min), 1.1 × 10−4 s−1 (t1/2 = 105 min), 1.0 × 10−3 s−1 (t1/2 = 11.5 min) and 4.5 × 10−4 s−1 (t1/2 = 26 min) in FBH The use of lower temperature (4 °C) for the determination of fu,brain in this study is not successful due to the instability of the compounds during longer equilibration times required at lower temperatures. The fu,brain values for a set of 15 CNS drugs determined in FBH and IBH-5 using HT-dialysis were similar and are consistent with the literature values. The use of IBH-5 led to the determination of fu,brain for unstable compounds that could not be determined by other methods. The use of IBH-5 is an easy and convenient method to determine the fu,brain of unstable compounds in FBH during drug discovery and development. [ABSTRACT FROM AUTHOR]

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]

• Development of a method allowing quantification of 10 UT and 3 precursors. • Quantification of total and free (protein unbound) UT. • Comparison between ultrafiltration and equilibrium dialysis for quantification of unbound UT. The objectives of this study were (1) to develop a method for the determination of 10 uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, kynurenine, p-cresyl glucuronide, p-cresyl sulfate, phenylacetylglutamine and trimethylamine N-oxide (TMAO)), and 3 precursors (tyrosine, phenylalanine, tryptophan) in serum and (2) to compare two separation methods to determine the free serum fraction: rapid equilibrium dialysis (RED) and ultrafiltration (UF). The method was developed on a liquid chromatography system coupled to a tandem mass spectrometer. Fifty µL of serum sample were precipitated with methanol after addition of internal standard. The two separation methods were compared using serum samples from patients suffering from renal impairment (n = 30). The method has been validated according to the European Medicines Agency (EMA) guidelines. Calibration curves were linear from 1 to 50 ng/mL up to 10,000–50,000 ng/mL according to the compounds. The comparison between the two separation methods produced similar results for all compounds except kynurenine, tryptophan (around 30% more with UF) and indole-3-acetic acid (around 30% more with RED). This study has allowed the development and validation of a sensitive and robust assay for the quantification of free and total concentrations of 10 uremic toxins and 3 precursors in human serum. [ABSTRACT FROM AUTHOR]

Fraction unbound (fu) values obtained from liver or hepatocyte homogenate with equilibrium dialysis (fu,homo) or the hepatocyte partition coefficient method at 4°C (fu,c) are both frequently used to estimate unbound drug concentrations (Cu) and unbound partition coefficient (Kpuu) of the liver. Literature data are somewhat controversial on this topic: some reported that the two methods gave comparable fu values, while others showed that they had no correlation. To better understand the two approaches, 44 structurally diverse compounds with wide ranges of fu values were used for the comparison study. The results indicate that fu values from the two methods are comparable with an average fold error of 2.9-fold and a bias of 1.0. Although some outliers were observed, the reasons are not entirely clear and further investigations are needed. As the fu data from both methods are correlated, fu,homo measurement using tissue homogenate is recommended over cells at 4°C (fu,c) in early drug discovery. This is because fu,homo method is more reliable, has good in vivo predictability, and feasibility for any tissue types where representative cells may not be readily available. The approach can be used to estimate Cu and Kpuu of tissues in order to develop pharmacokinetic/pharmacodynamic relationships, and to estimate therapeutic indices, as well as to predict drug-drug interactions. [ABSTRACT FROM AUTHOR]

The present study showed the binding interaction of 2-(4,5-Dihydro-1,2-oxazol-5-yl)-phenol-N-methylaniline (2DHOPNA) with BSA in 1,4-dioxane, DMSO and DMF by equilibrium dialysis, FT-IR, acoustical at physiological pH and its molecular modeling study. Findings were interpreted by scatchard plot which showed an increase in association constants with increasing temperature and concentrations of the 2DHOPNA. It is seen that the binding supposed to be more significant in 1,4-dioxane than DMSO and DMF. Thermodynamic parameters are also determined for the binding interaction of 2DHOPNA with BSA. Values of Gibb's free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) were calculated by using van't Hoff equation. The positive values of ΔH and ΔS showed exothermic interaction between 2DHOPNA and BSA. Similarly, negative ΔG showed the spontaneity of the binding process. ΔG becomes more negative with increase in temperature, indicated feasibility of the binding interaction at high temperature. Molecular modeling confirmed the binding interaction having energy of -210.13. [ABSTRACT FROM AUTHOR]

The metal ions play a vital role in a large number of widely differing biological processes. Some of these processes are quite specific in their metal ion requirements. In that only certain metal ions, in specific oxidation states, can fulfil the necessary catalytic or structural requirement, while other processes are much less specific. In this paper, the interactions between triethanolamine, lauryl sulphate and albumin molecules have been reported. The pH and diffusion current measurements on the binding of copper and mercury ions with albumin have been discussed. The effect of physico-chemical factors on the interaction between divalent metal ions and albumin has been carried out. Physico-chemical studies on the binding of Hg(II) and Cu (II) with albumin have been discussed. [ABSTRACT FROM AUTHOR]

Moxidectin (MOX) is a macrocyclic lactone used to eliminate endo and ectoparasites in many mammalian species. It is notably the active ingredient of the anti-parasitic drug Cydectin®, manufactured by Virbac, and is frequently used to treat sarcoptic mange in Australian wildlife. Protein binding plays a significant role in the efficacy of a drug, as the unbound/free drug in plasma ultimately reflects the pharmacologically relevant concentration. This study aimed to investigate the free drug percentage of Moxidectin after in vitro spiking into the sera of four sarcoptic mange-susceptible Australian wildlife species; the koala (Phascolarctos cinereus), the bare-nosed wombat (Vombatus ursinus), the eastern grey kangaroo (Macropus giganteus), and the mountain brushtail possum (Trichosurus cunninghami). Three concentration points of MOX were tested for each individual: 20 pg/μL, 100 pg/μL and 500 pg/μL. Serum from five individuals of each species underwent an equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed an atypical concentration dependent binding across all species, where free drug percentage decreased as MOX concentration increased. In addition, wombats showed significantly lower free drug levels. These findings call for further research into the mechanisms of moxidectin protein binding to help understand MOX pharmacokinetics in marsupials. [Display omitted] • The concentration dependent binding of MOX to plasma proteins in serum is atypical in the koala (Phascolarctos cinereus), the bare-nosed wombat (Vombatus ursinus), the eastern grey kangaroo (Macropus giganteus), and the mountain brushtail possum (Trichosurus cunninghami). • Bare-nosed wombats have lower free drug levels of MOX compared to other Australian marsupial species. • Interspecies differences in vitro MOX indicates species specific treatment dosages may need to be developed. • Further research needs to be conducted to establish the reason behind this novel pattern of protein binding, particularly in bare-nosed wombats. [ABSTRACT FROM AUTHOR]

Platinum (Pt) antineoplastic agents remain indispensable for the treatment of oncological disease. Pt-based drugs are mainly used in the therapy of ovarian cancer and non-small-cell lung carcinoma. A novel platinum-containing antineoplastic agent BP-C1 is a complex of diamminoplatinum with an oxygen-donor polymeric ligand of benzene-polycarboxylic acids, isolated from natural lignin. The aim of the study was to investigate ex vivo protein binding of BP-C1. Protein binding of BP-C1 was tested using equilibrium dialysis. Pooled blood plasma was used in the study. Control solutions contained the same dosages of BP-C1 in PBS (pH 7.2). Plasma and control solutions were submitted to equilibrium dialysis across a vertical 8 kDa cut-off membrane for 4 h at 37 °C under gentle shaking. Platinum was quantified in dialysis and retained fractions using inductively coupled plasma mass spectrometry after microwave digestion. The dialysis system was tested and validated; this showed no protein saturation with platinum. A medium degree of binding of platinum to macromolecular species of ca. 60% was observed. The study showed the maintenance of a high fraction of free BP-C1 in the bloodstream, facilitating its pharmacological activity.