Saturation Of Protein Binding Research Articles

Serum proteins on nanoparticles: early stages of the "protein corona".

Nanoparticles in biological systems such as the bloodstream are exposed to a complex solution of biomolecules. A "corona" monolayer of proteins has historically been thought to form on nanoparticles upon introduction into such environments. To examine the first steps of protein binding, Fluorescence Correlation/Cross Correlation Spectroscopy and Fluorescence Resonance Energy Transfer were used to directly analyze four different nanoparticle systems. CdSe/ZnS core/shell quantum dots, 100 nm diameter polystyrene fluospheres, 200 nm diameter polystyrene fluospheres, and 200 nm diameter PEG-grafted DOTAP liposomes were studied with respect to serum protein binding, using bovine serum albumin as a model. Surface heterogeneity is found to be a key factor in protein binding to these nanoparticles, and as such we present a novel conceptualization of the early hard corona as low-ratio, non-uniform binding rather than a uniform monolayer.

Physiologically Based Pharmacokinetic Modeling of Salivary Concentrations for Noninvasive Biomonitoring of 2,4-Dichlorophenoxyacetic Acid (2,4-D).

Saliva has become a favorable sample matrix for biomonitoring due to its noninvasive attributes and overall flexibility in collection. To ensure measured salivary concentrations reflect the exposure, a solid understanding of the salivary transport mechanism and relationships between salivary concentrations and other monitored matrices (ie, blood, urine) is needed. Salivary transport of a commonly applied herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was observed in vitro and in vivo and a physiologically based pharmacokinetic (PBPK) model was developed to translate observations from the cell culture model to those in animal models and further evaluate 2,4-D kinetics in humans. Although apparent differences in experimental in vitro and in vivo saliva:plasma ratios (0.034 and 0.0079) were observed, simulations with the PBPK model demonstrated dynamic time and dose-dependent saliva:plasma ratios, elucidating key mechanisms affecting salivary transport. The model suggested that 2,4-D exhibited diffusion-limited transport to saliva and was additionally impacted by protein binding saturation and permeability across the salivary gland. Consideration of sampling times post-exposure and potential saturation of transport mechanisms are then critical aspects for interpreting salivary 2,4-D biomonitoring observations. This work utilized PBPK modeling in in vitro to in vivo translation to explore benefits and limitations of salivary analysis for occupational biomonitoring.

Population-based meta-analysis of roxithromycin pharmacokinetics: dosing implications of saturable absorption and protein binding.

The macrolide antibiotic roxithromycin has seen widespread clinical use for several decades; however, no population pharmacokinetic analysis has been published. Early studies indicated saturation of protein binding and absorption at doses within the approved range, which may impact pharmacodynamic target attainment since regimens of 150 mg twice daily and 300 mg once daily are used interchangeably in clinical practice. This study aimed to develop a population-based meta-analysis of roxithromycin pharmacokinetics, and utilize this model to inform optimal dosing regimens. Following an extensive search, roxithromycin pharmacokinetic data were collected or digitized from literature publications. Population pharmacokinetic modelling was undertaken with ADAPT. Dosing simulations were performed to investigate differences in exposure and pharmacodynamic target attainment between dosing regimens. A two-compartment model with saturable absorption described the data ( n = 63); changes in free drug exposure were simulated using a saturable protein binding model. Simulations indicated that a 300 mg daily regimen achieves a 37% and 53% lower total or free AUC ( f AUC), respectively, compared with 150 mg twice daily. These pharmacokinetic differences translated to significantly lower target attainment ( f AUC/MIC ratio >20) with a 300 mg daily regimen at MICs of 0.5 and 1 mg/L (51% and 7%) compared with patients receiving 150 mg twice daily (82% and 54%). Roxithromycin displays saturable absorption and protein binding leading to lower exposure and lower target attainment at MICs ≥0.5 mg/L with widely used once-daily dosing regimens, indicating that twice-daily regimens may be preferable for pathogens less susceptible to roxithromycin.

Mechanistic investigations into the species differences in pinometostat clearance: impact of binding to alpha-1-acid glycoprotein and permeability-limited hepatic uptake

1. The plasma clearance of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was shown to be markedly lower in human compared to the preclinical species, mouse, rat and dog.2. This led to vertical allometry where various interspecies scaling methods were applied to the data, with fold-errors between 4 and 13. We had previously reported the elimination and metabolic pathways of EPZ-5676 were similar across species. Therefore, the aim of this work was to explore the mechanistic basis for the species difference in clearance for EPZ-5676, focusing on other aspects of disposition.3. The protein binding of EPZ-5676 in human plasma demonstrated a non-linear relationship suggesting saturable binding at physiologically relevant concentrations. Saturation of protein binding was not observed in plasma from preclinical species. Kinetic determinations using purified serum albumin and alpha-1-acid glycoprotein (AAG) confirmed that EPZ-5676 is a high affinity ligand for AAG with a dissociation constant (Kd) of 0.24 μM.4. Permeability limited uptake was also considered since hepatocyte CLint was much lower in human relative to preclinical species. Passive unbound CLint for EPZ-5676 was estimated using a correlation analysis of logD and data previously reported on seven drugs in sandwich cultured human hepatocytes.5. Incorporation of AAG binding and permeability limited hepatic uptake into the well-stirred liver model gave rise to a predicted clearance for EPZ-5676 within 2-fold of the observed value of 1.4 mL min−1 kg−1. This analysis suggests that the marked species difference in EPZ-5676 clearance is driven by high affinity binding to human AAG as well as species-specific hepatic uptake invoking the role of transporters.

A population pharmacokinetic model of valproic acid in pediatric patients with epilepsy: a non-linear pharmacokinetic model based on protein-binding saturation.

Valproic acid (VPA) follows a non-linear pharmacokinetic profile in terms of protein-binding saturation. The total daily dose regarding VPA clearance is a simple power function, which may partially explain the non-linearity of the pharmacokinetic profile; however, it may be confounded by the therapeutic drug monitoring effect. The aim of this study was to develop a population pharmacokinetic model for VPA based on protein-binding saturation in pediatric patients with epilepsy. A total of 1,107 VPA serum trough concentrations at steady state were collected from 902 epileptic pediatric patients aged from 3weeks to 14years at three hospitals. The population pharmacokinetic model was developed using NONMEM(®) software. The ability of three candidate models (the simple power exponent model, the dose-dependent maximum effect [DDE] model, and the protein-binding model) to describe the non-linear pharmacokinetic profile of VPA was investigated, and potential covariates were screened using a stepwise approach. Bootstrap, normalized prediction distribution errors and external evaluations from two independent studies were performed to determine the stability and predictive performance of the candidate models. The age-dependent exponent model described the effects of body weight and age on the clearance well. Co-medication with carbamazepine was identified as a significant covariate. The DDE model best fitted the aim of this study, although there were no obvious differences in the predictive performances. The condition number was less than 500, and the precision of the parameter estimates was less than 30%, indicating stability and validity of the final model. The DDE model successfully described the non-linear pharmacokinetics of VPA. Furthermore, the proposed population pharmacokinetic model of VPA can be used to design rational dosage regimens to achieve desirable serum concentrations.

The Impact of Dosage of Sustained-Release Formulation on Valproate Clearance and Plasma Concentration in Psychiatric Patients

Pharmacokinetic parameters were compared between 2 dosages of valproic acid (VPA) sustained-release (SR) formulation in psychiatric patients. A total of 66 psychiatric patients (21 women and 45 men; age range, 18-60 years) receiving 500 mg/d (n = 28) or 1000 mg/d (n = 38) of VPA SR for at least 4 weeks were recruited into the study. A 5-mL blood sample was collected into a plain tube and immediately centrifuged for plasma. Separation of free VPA was further done using Centrifree micropartition devices. Both total and free VPA concentrations (C(total) and C(free)) were analyzed by TDx analyzer with fluorescence polarization immunoassay technique. The patients' characteristics and pharmacokinetic parameters were compared between the 2 dosage groups using independent t test or χ² test where appropriate. The results show that the increment in C(total) (mg/mL) for every milligram-per-kilogram increment in dosage was decreased from 7 ± 3 to 4 ± 1 (mg/L) / (mg/kg) when the total VPA clearance (CL(total)) increased from 6.1 ± 2.6 to 9.0 ± 3.1 mL/kg per hour with the increasing dose in the 500 mg/d and 1000 mg/d groups, respectively (P < 0.05). The increment in C(free) [0.6 ± 0.3 vs 0.5 ± 0.2 (mg/L) / (mg/kg)] and CL(free) (80.4 ± 41.5 vs 92.2 ± 47.6 ml/kg per hour) were not significantly different. Owing to the saturation of protein binding, percent free VPA was significantly increased from 8 ± 3 to 11 ± 3 when the dose was increased from 500 to 1000 mg/d (P < 0.05). In conclusion, an increase in the VPA dose resulted in a disproportional increase between dosage and C(total), whereas a proportional increase between dosage and C(free) still existed. Therefore, our study suggests that the therapeutic range of C(free) is 4 to 12 mg/L based on the therapeutic range of C(total) (45-100 mg/L) for general psychiatric conditions.

Severe valproic acid intoxication: case study on the unbound fraction and the applicability of extracorporeal elimination

Among anticonvulsants, valproic acid (VPA) is cited as the most frequent cause of unintentional and intentional intoxications. Symptoms of VPA intoxication are diverse and are related to VPA plasma concentration. Although total plasma concentrations of less than 450 mg/l produce limited toxicity, severe intoxications (>850 mg/l) can induce coma and are ultimately life threatening. A 32-year-old comatose woman was admitted to the ICU at our hospital; she suffered from hypotension, respiratory depression, hypoglycaemia, sinus bradycardia, hyperammonaemia, metabolic acidosis, and her core body temperature was 33.7 degrees C. The total VPA plasma concentration was 1244 mg/l with an increased unbound fraction of 85%. After we administered multiple doses of activated charcoal, she underwent continuous veno-venous haemofiltration to reduce the plasma VPA concentration. As the total concentration decreased, the unbound fraction also decreased. Within 20 h of admission, the patient made a full recovery. In cases of VPA intoxication, protein-binding saturation and drug characteristics render extracorporeal elimination, an effective technique for eliminating the unbound drug. Its application should be considered, depending on clinical symptoms, VPA concentration (>300 mg/l), albumin concentration and ammonia concentration (>400 micromol/l). The application of this technique should be weighed against its risks. This case illustrates the clinical significance of applying continuous veno-venous haemofiltration in VPA intoxication because of protein-binding saturation, and suggests when extracorporeal elimination should be considered.

Pharmacokinetics of YK754, a novel If channel inhibitor in rats, dogs and humans

The pharmacokinetics of YM758, a novel funny If current channel (If channel) inhibitor, was investigated after single intravenous (i.v.) and oral dosing to rats and dogs, and partially compared with the results in humans by using liver microsomes. After i.v. administration, the plasma YM758 concentrations decreased, with an elimination half-life (t(1/2)) of 1.14-1.16 h in rats and 1.10-1.30 h in dogs. Total body clearance (CL(tot)) was 5.71-7.27 and 1.75-1.90 L/h/kg in rats and dogs, respectively which was comparable to the hepatic blood flow rate. In dogs, the pharmacokinetic profiles for i.v. bolus administration and continuous infusion did not differ. After oral administration, the levels of YM758 in rat plasma increased more than dose-proportionally, whereas almost linear pharmacokinetics were observed in dogs. Absolute bioavailability was 7.5%-16.6% in rats and 16.1%-22.0% in dogs. The plasma protein binding saturation of YM758 was observed in dogs and humans; this finding is consistent with the result that the major binding protein of YM758 in plasma is alpha1-acid glycoprotein (AGP), in particular in humans. The blood-to-plasma partition coefficient values were 1.36-1.42 in rats, 0.95-1.15 in dogs and 0.71-0.85 in humans. The results of the metabolic study on liver microsomes indicated that the non-linear pharmacokinetics of YM758 observed in rats may be partially due to a first-pass effect in the gastrointestinal tract and the liver.

Toxicokintic and toxicodynamic analysis of clofibrate based on free drug concentrations in nagase analbuminemia rats (NAR)

Toxicokinetics (TK) is usually performed by measurement of the total drug concentrations in plasma. However, free drug concentrations in plasma are considered to correlate directly with toxicodynamics (TD). In the present study, to evaluate the applicability of TK/TD analysis based on free drug concentrations, we investigated the TK/TD of clofibrate, which binds to albumin with a higher ratio, using an albumin-deficient mutant strain, Nagase analbuminemia rats (NAR). TK, blood chemistry, histopathology, drug and fatty acid metabolizing enzymes and microarray analysis in the liver were examined after a 4-day oral administration of clofibrate. Compared to Sprague-Dawley (SD) rats, the parent strain of NAR, 4.1-fold higher AUC(0-24hr) based on free drug concentrations (3445 versus 844 microg.hr/ml) was observed in NAR when both rats showed the same level of AUC(0-24hr) based on the total drug concentrations (4436 versus 4237microg.hr/ml). Additionally, more severe hepatocellular hypertrophy, increase in aspartate transaminase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), decrease in total cholesterol (T.CHO), phospholipid (PL), triglyceride (TG), and non-esterified fatty acid (NEFA), and increase in the mRNA levels of fatty acid metabolizing enzymes (FAOS, CAT, and CPT) were observed in NAR at the same dose. These results demonstrated that NAR developed more severe toxicities and pharmacological effects than SD rats correlating with the higher AUC of the free drug concentrations. The results also suggested that TK/TD analysis based on the free drug concentration is appropriate to interpret the relationship between exposure and toxicity in cases of protein binding saturation including protein decrease or species differences on protein binding, especially when drugs showing a higher protein binding ratio are dosed.

Pharmacokinetic Considerations in Clinical Toxicology

Pharmacokinetic and pharmacodynamic principles should be regarded in the assessment and proper management of patients exposed to a poison. Clinicians must apply these principles to make rational clinical decisions regarding the significance of the poisoning (risk assessment) and to formulate an appropriate management plan. However, pharmacokinetic processes and parameters may be changed in the patient with acute poisoning. This may result from saturation of the capacity of a number of physiological processes due to the high dose, or the toxic effects of the poison may change these processes directly. For example, absorption kinetics may be altered because of increased gastrointestinal transit time (e.g. cholinergic receptor antagonists) or saturable absorption (e.g. methotrexate). Saturation of protein binding may increase the volume of distribution and thereby increase the elimination half-life (e.g. salicylates). Alteration of the acid-base balance (poison-induced or iatrogenic) may also increase or decrease the distribution of a poison. Saturation of metabolism at high doses can prolong toxicity (e.g. phenytoin) or lead to other routes of metabolism that lead to increased toxicity (e.g. paracetamol [acetaminophen]). Excretion may be reduced by saturation of active transporters or decreased renal blood flow.A better understanding of pharmacokinetic principles should improve the clinical care of patients. It should lead to more accurate interpretation of blood concentrations or biomarkers (e.g. ECG intervals or acetylcholinesterase activity) and how these relate to the time course for that poison, and better prediction of prognosis. This in turn, indicates the appropriate duration of observation and the requirement for some specific treatments. Many specific poisoning treatments aim to favourably alter the pharmacokinetics of the poison. These include activated charcoal, whole bowel irrigation, extracorporeal elimination, chelating agents, antitoxins and urinary alkalinisation. The evidence supporting them, their indications and limitations can only be understood using pharmacokinetic principles. These principles also underpin the appropriate choice within the flexible dosage regimen for many antidotes. In particular, naloxone, flumazenil, methylene blue, atropine and pralidoxime all use variable doses and have an elimination half-life that is much shorter than many (but not all) of the poisons treated by these agents. A firm grounding in pharmacokinetics/toxicokinetics should be regarded as a core competency for all professionals involved in clinical care or undertaking research in clinical toxicology.

Microdialysis in Drug Discovery

Microdialysis has been developed during the last 25 years by several authors primarily to study brain function and changes in levels of endogenous compounds such as neurotransmitters or metabolites. The development of microdialysis for the purpose of measuring drugs was initiated during the late eighties. This technique provides a means of continuous plasma sampling without repeated blood sampling and the applicability to the study of drug metabolism and pharmacokinetics in experimental animals and human. Also, the microdialysis technique allows the study of plasma protein binding and the saturation of protein binding. The implantation of the microdialysis probe in other tissues and organs, like central nervous system, adipose tissue and heart, allows the study of drug distribution. On the other hand, the measurement of endogenous substances using the microdialysis technique permits the study of the effect of drugs on neurotransmission and metabolism. Moreover, as this technique allows the simultaneous determination of different physiological parameters such as blood pressure, locomotor and convulsive activity, it is a suitable tool for pharmacokinetic-pharmacodynamic studies of drugs and pharmacokinetic-pharmacodynamic (PK-PD) modeling. Lastly, the reverse microdialysis is a powerful technique for the study of local actions of drugs in different tissues such as specific brain nuclei, myocardium, liver or skeletal muscle. So, this article reviewed the vast applications of the microdialysis technique for the study of pharmacokinetic and pharmacodynamic properties of drugs.

Dose‐dependent plasma clearance of MK‐826, a carbapenem antibiotic, arising from concentration‐dependent plasma protein binding in rats and monkeys

After intravenous administration of MK‐826, a new carbapenem antibiotic, the compound exhibited nonlinear pharmaco‐kinetics in rats and monkeys. In both species, time‐averaged plasma clearance (based on total concentrations) increased about 5‐fold over the 10‐ to 180‐mg/kg dose range. MK‐826 was extensively plasma protein bound in rat and monkey plasma, and the extent of binding was concentration dependent at plasma concentrations achieved after administration of these doses. Rosenthal analysis of the plasma protein binding indicated that there were two classes of binding sites. The binding capacity of the primary site was comparable to the plasma albumin concentration, which suggested that this primary site consisted of a single site on albumin. The extent of binding of MK‐826 to rat albumin was similar to that in whole plasma. Clearance values based on unbound concentrations appeared independent of dose from 10 to 180 mg/kg, which is consistent with saturation of protein binding as the primary cause of the nonlinear pharmacokinetic behavior.

Clinical pharmacokinetics of nabumetone. The dawn of selective cyclo-oxygenase-2 inhibition?

Nabumetone is a nonsteroidal anti-inflammatory drug (NSAID) of the 2,6-disubstituted naphthyl-alkanone class. Nabumetone is metabolised to an active metabolite 6-methoxy-2-napthylacetic acid (6-MNA) which is a relatively selective cyclo-oxygenase-2 inhibitor that has anti-inflammatory and analgesic properties. Nabumetone and its metabolites bind extensively to plasma albumin. Nabumetone is eliminated following biotransformation to 6-MNA, which does not undergo enterohepatic circulation and the respective glucoroconjugated metabolites are excreted in urine. Substantial concentrations of 6-MNA are attained in synovial fluid, which is he proposed site of action in chronic inflammatory arthropathies. A smaller area under the plasma concentration-time curve (AUC) is evident at steady state as compared with a single dose; this is possibly due to an increase in the volume of distribution and saturation of protein binding. Relationships between 6-MNA concentrations and the therapeutic and toxicological effects have yet to be elucidated for this NSAID. Renal failure significantly reduces 6-MNA elimination but steady-state concentrations of 6-MNA are not increased, possibly because of nonlinear protein binding. Elderly patients with osteoarthritis demonstrate decreased elimination and increased plasma concentrations of nabumetone as compared with young healthy volunteers. Rheumatic disease activity also influences 6-MNA plasma concentrations, as patients with more active disease and lower serum albumin concentrations demonstrate a lower area under the plasma concentration versus time curve. A reduced bioavailability of 6-MNA in patients with severe hepatic impairment is also evident. Dosage adjustment may be required in the elderly, patients with active rheumatic disease and those with hepatic impairment, but not in patients with mild-to-moderate renal failure.

In vitro study of the protein binding of fusidic acid: A contribution to the comprehension of its pharmacokinetic behaviour

Fusidic acid (FA: Fucidine® Leo Laboratories) is a type I drug: acid, ionized at plasma pH, with a high intrinsic affinity for albumin. In the case of this kind of product, the possibility exists of the saturation of protein binding and of drug interactions. The relative affinity of fusidate (F) for different solutions of albumin was compared in vitro, i.e., purified industrial albumin, fresh frozen plasma, and plasma from surgical intensive care unit (ICU) patients. The theoretical consequences of the albumin status of a patient on FA kinetics were envisaged in the form of a model. The three stock solutions were diluted such that solution S1 yielded concentrations of 38, 29 and 19 g/l of albumin, and S2 provided concentrations of 44, 32 and 20 g/l of albumin. The concentration of solution S3 was initially 24 g/l (severe hypoalbuminemia). The seven solutions were spiked with known quantities of FA (10, 50, 100 and 150 mg/l) in which total F (tF) and free F (fuF) were assayed by HPLC. The per cent binding of F to albumin was determined, as was the number of binding sites (n) and its saturable or non-saturable character as a function of the medium (Scatchard plots). The conclusions drawn were: (1) the number of F binding sites on albumin is between two and three and the per cent binding of F is in the range of 91.5–98.7%; (2) Fucidine® belongs to the group of drugs sensitive to the albumin status of the media in which it is added; (3) the often severe hypoalbuminemia of ICU patients may lead to a considerable increase in the active fraction fuF; the most seriously affected patients should be more exposed to variations in the tF/fuF ratio; (4) the clinical status of the patients (kidney failure, malnutrition hypoalbuminemia, etc.) should be taken into account when determining the dosage schedules of parenterally administered FA. In summary, after repeated administration, the significant increase in fuF should induce the following: an increase in Vd, an increase then a decrease in Cl, a decrease then an increase in the AUC. The hypothesis according to which Fucidine® would be more effective in the hypoalbuminemic patient remains to be confirmed in selected patients with coupled measurements of tF anf fuF.

てんかん患者の遊離型バルプロ酸濃度に及ぼす血清中アルブミンと年齢の影響

The serum protein binding of valproic acid (VPA) was examined with samples collected front 88 epileptic patients whose ages ranged from 3 to 78 years.Blood samples were usually collected within one to six hours after a dose in the morning.The free fraction of VPA was obtained with a microultrafiltration devices (Molcut II®) which was developed by Nihon Millipore Ltd., Tokyo, Japan.The free and total VPA levels in serum were determined by fluorescence polarization immunoassay (TDX®).The serum albumin levels were determined by the procedure of ionophoresis.Total VPA levels were positively correlated with free fraction (Y=0.13X+6.39, n=88, r=0.67), and a negative correlation between serum albumin levels and free fraction of VPA (Y=-6.99X+48.49, n=88, r=0.54) was observed in the epileptic patients.We found that the serum albumin levels were decreased with the aging, and the total VPA levels were higher in younger children.The increases of free fraction in the children (8 patients in Lennox Syndrom) and the elderly (11 patients in Hypoalbuminemia) might be results from the protein binding saturation.

Gamma globulin protection of radioimmunoassay and competitive protein binding saturation analysis of steroids.

In spite of careful distillation and purification of solvents and materials used for extraction and chromatography in steroid radioimmunoassays and competitive protein binding assays, it was impossible to eliminate blank values. Since dried down solvent residues effected the activity of the binding proteins it was necessary to protect the proteins against these effects. By adding 100μl of an aqueous 10g% γ-globulin solution to each assay tube a dilution of the small amount of binding protein in a large amount of γ-globulin was achieved. The huge excess of protein served to protect the binding proteins against solvent residue effects and thus to eliminate the blank value in our assays.