Nonlinear Plasma Protein Binding Research Articles

Physiologically-based pharmacokinetic model for 2,4-dinitrophenol.

New approaches in drug development are needed to address the growing epidemic of obesity as the prevalence of obesity increases worldwide. 2,4-Dinitrophenol (DNP) is an oxidative phosphorylation uncoupling agent that was widely used in the early 1930s for weight loss but was quickly banned by the FDA due to the severe toxicities associated with the compound. One of the limitations leading to the demise of DNP as a pharmaceutical was a lack of understanding about the pharmacokinetic-pharmacodynamic relationship. The purpose of this study was to investigate whole body disposition of DNP in order to understand the relationship between the pharmacokinetics, efficacy and toxicity in the C57BL/6J diet induced obese mouse model. Following intravenous administration of 1mg/kg, and intraperitoneal administration of 5mg/kg and 15mg/kg of DNP, we found limited DNP distribution to tissues. Experimentally measured partition coefficients were found to be less than 1 for all analyzed tissues. In addition, DNP exhibits significant nonlinear pharmacokinetics, which we have attributed to nonlinear plasma protein binding and nonlinear partitioning into liver and kidney. By enhancing our understanding of the PK-PD relationship, we can develop new approaches to leverage oxidative phosphorylation uncoupling as a weight loss strategy.

Population Pharmacokinetics of Unbound and Total Teicoplanin in Critically Ill Pediatric Patients.

Teicoplanin is a highly protein-bound antibiotic, increasingly used to treat serious Gram-positive infections in critically ill children. Maturational and pathophysiological intensive care unit-related changes often lead to altered pharmacokinetics. In this study, the objectives were to develop a pediatric population-pharmacokinetic model of unbound and total teicoplanin concentrations, to investigate the impact of plasma albumin levels and renal function on teicoplanin pharmacokinetics, and to evaluate the efficacy of the current weight-based dosing regimen. An observational pharmacokinetic study was performed and blood samples were collected for quantification of unbound and total concentrations of teicoplanin after the first dose and in assumed steady-state conditions. A population-pharmacokinetic analysis was conducted using a standard sequential approach and Monte Carlo simulations were performed for a probability of target attainment analysis using previously published pharmacokinetic-pharmacodynamic targets. A two-compartment model with allometric scaling of pharmacokinetic parameters and non-linear plasma protein binding best described the data. Neither the inclusion of albumin nor the renal function significantly improved the model and no other covariates were supported for inclusion in the final model. The probability of target attainment analysis showed that the standard dosing regimen does not satisfactory attain the majority of the proposed targets. We successfully characterized the pharmacokinetics of unbound and total teicoplanin in critically ill pediatric patients. The highly variable unbound fraction of teicoplanin could not be predicted using albumin levels, which may support the use of therapeutic drug monitoring of unbound concentrations. Poor target attainment was shown for the most commonly used dosing regimen, regardless of the pharmacokinetic-pharmacodynamic target evaluated.

Physiologically Based Pharmacokinetic Model of the DPP-4 Inhibitor Linagliptin to Describe its Nonlinear Pharmacokinetics in Humans

Linagliptin, a dipeptidyl peptidase (DPP)-4 inhibitor, for type 2 diabetes exhibits nonlinear plasma protein binding in the therapeutic concentration range due to its high affinity binding to the pharmacological target DPP-4, and its pharmacokinetics both in plasma and urine is also nonlinear. The purpose of the present study was to explain the nonlinear pharmacokinetic profiles using a physiologically based pharmacokinetic (PBPK) model with saturable binding of linagliptin to soluble and membrane-bound DPP-4 in blood and organs including kidneys. The model was first fitted to previously reported full-scale plasma concentrations and urinary excretion data at 4 intravenous (iv) dose levels. Additional fitting to the data from 4 oral (po) dose levels was then performed to yield the final iv-po based model including gastrointestinal absorption-associated parameters. Data from [14C]linagliptin mass balance study were also used for optimizing parameters related to enterohepatic circulation. The PBPK model was thus constructed and well describes the nonlinear pharmacokinetic profiles of linagliptin in both plasma and urine, demonstrating that the nonlinear pharmacokinetics are fully explained by its specific binding to target protein. The present study thus introduces the involvement of target-mediated disposition for linagliptin in humans.

Lefamulin: novel pleuromutilin drug for community acquired bacterial pneumonia

The advent and spread of antimicrobial resistance has led to a global public health emergency necessitating development of new antimicrobial drugs. Community acquired bacterial pneumonia (CABP) contributes a major portion of societal burden with increasing morbidity due to evolution of drug resistant strains. Lefamulin is a novel pleuromutilin antibiotic with unique mechanism of action through inhibition of protein synthesis by binding to the peptidyl transferase center of the 50s bacterial ribosome. The drug displays activity against Gram positive and atypical organisms associated with CABP (i.e., Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumonia, Legionella pneumophila, and Chlamydophila pneumoniae), with an expanded Gram-positive spectrum including Staphylococcus aureus (i.e., methicillin-resistant, vancomycin-intermediate, and heterogeneous strains). Lefamulin is available in both intravenous (IV) and per oral (PO) formulation, exhibits high nonlinear plasma protein binding with low unbound concentrations, higher concentrations in lung epithelial lining fluid (ELF) than in plasma, and a half-life of approximately 10 hour. The recommended IV dose is 150 mg twice daily over 1 hour or a PO dose of 600 mg twice daily. Most common adverse drug reactions injection site reactions, hepatic enzyme elevation, nausea, diarrhoea, hypokalemia, insomnia, and headache. Clinical trials for lefamulin have been positive and Phase 3 data suggest similar efficacy when compared to moxifloxacin with or without linezolid in CABP. Also, the documented resistance and cross-resistance with other Gram-positive antibacterials remains low. With Nabrivia Pharmaceuticals having already received US FDA approval in August 2019, lefamulin may soon be a new addition to the mounting armoury of drugs against CABP. 

Concentration-dependent plasma protein binding: Expect the unexpected

The unbound fraction of a drug in plasma can profoundly influence both its pharmacokinetics and pharmacodynamics. For most drugs, the unbound fraction is relatively constant across the clinically relevant range of concentrations in a given individual. Nonlinear plasma protein binding involving saturation of binding sites results in increasing unbound fraction as the concentration of the drug increases, a phenomenon that is consistent with the law of mass action and is well recognized. Not widely appreciated is that some drugs undergo atypical concentration-dependent binding to plasma proteins, whereby the unbound fraction decreases with increasing concentration. In this article we review the drugs for which atypical nonlinear plasma protein binding has been reported. For each drug, the evidence for the phenomenon is presented and the proposed mechanism discussed. Also reviewed are the potential implications of atypical nonlinearity in plasma protein binding. Highlighted is the importance of understanding the relationship between unbound fraction and plasma drug concentration during the preclinical and early clinical stages of drug development, and during the routine clinical use of a drug especially if therapeutic drug monitoring is used to assist in optimization of the dosing regimen. The lesson is that the unexpected concentration-dependent behavior that has been observed for a number of drugs should be expected to occur for some other drugs.

A need to revisit clinical breakpoints of tigecycline: effect of atypical non-linear plasma protein binding

Tigecycline is highly active against various drug-resistant bacteria. The US Food and Drug Administration (FDA) recently issued a black box warning for tigecycline owing to an associated increase in all-cause mortality. Clinical breakpoints of antibiotics are vital in susceptibility testing of pathogens for the selection of antibiotic therapy; however, no consensus exists between different committees on the clinical breakpoints of tigecycline. Of note, tigecycline exhibits atypical non-linear plasma protein binding (PPB) behaviour, and the pivotal probability of target attainment (PTA) analysis for the determination of clinical breakpoints did not account for the PPB of tigecycline. In this work, the PTA analysis was performed with consideration of atypical non-linear PPB behaviour of tigecycline. A model describing atypical non-linear PPB was developed and validated. Monte Carlo simulations were performed to determine the target ratio of area under the free drug concentration–time curve to minimum inhibitory concentration (fAUC/MIC) for Escherichia coli and, subsequently, PTA analyses were performed. The target fAUC/MIC ratio for E. coli was determined as 2.05, whilst the target AUC/MIC ratio was 6.96. The PTA analyses suggest a lower clinical breakpoint of tigecycline against E. coli. This finding suggests that there is a need to revisit the current clinical breakpoints of tigecycline.

Role of Divalent Metal Ions in Atypical Nonlinear Plasma Protein Binding Behavior of Tigecycline

In typical nonlinear plasma protein binding (PPB) behavior, the free fraction increases with increasing total concentrations. In contrast, when a drug exhibits atypical nonlinear PPB behavior, the free fraction decreases with increasing total concentrations. Tigecycline, a novel glycylcycline, exhibits atypical nonlinear PPB behavior, but the mechanism of such behavior is currently unknown. Because tigecycline can form complexes with metal ions, an interaction between metal ion, tigecycline, and plasma proteins was hypothesized but not further investigated. The current work explores the role of metal ions in the atypical nonlinear PPB behavior of tigecycline and proposes a plausible mechanism of atypical nonlinear PPB behavior. The addition of ethylenediaminetetraacetic acid resulted in 10- to 30-fold higher unbound fractions, and the atypical behavior was nullified. The saturation of ethylenediaminetetraacetic acid chelation, by addition of excessive divalent metal ions, such as calcium and magnesium, led to the return of the atypical nonlinear PPB behavior. Different possible mechanisms were evaluated by simulation, and a plausible mechanism was proposed.

Antiepileptic Drugs: Adult

Abstract 1 Alaa M. Ahmad, 1 Frank D. Boudinot, 2 William H. Barr, 3 Ronald C. Reed, and 2 William R. Garnett ( 1 Pharmaceutics, Virginia Commonwealth University, Richmond, VA ; 2 Pharmacy, Virginia Commonwealth University, Richmond, VA ; and 3 Neuroscience, Abbott Laboratories Inc., Abbott Park, IL ) Rationale: Clinicians rely primarily on total valproic acid (VPA) concentrations for therapeutic monitoring. However, due to nonlinear plasma protein binding of VPA, dose titration is complicated by disproportionality between the total drug concentration and dose. The purpose of this report was to develop a nomogram for VPA based on total drug concentrations. Methods: Nine healthy volunteers were administered 500, 750 and 1000 mg VPA in a dose escalation study. Plasma protein binding of VPA was characterized for each volunteer in vitro. Results: A one-site saturable binding model provided an adequate description of the binding of VPA to albumin and yielded (mean,% standard error) 2.3 (4.5%) binding sites (N) and an equilibrium association constant (KA) of 6.7 (17.9%) (L/mM). Predictions for the increase in total concentration observed with dose escalation were based on individual and population pharmacokinetic parameters. There weren't significant statistical differences between the predictions based on individual and population estimates (based on root mean square error and mean absolute error). Conclusions: Using population estimates, a dosing nomogram for VPA was constructed. To minimize the risk of achieving toxic drug concentrations, the dose should not be increased more than 2 fold at a time. The nomogram should be used in conjunction with patient history and clinical response to aid clinicians in making informed decisions about dose-adjustments. The nomogram can be used for all VPA preparations and regardless of enzyme inducing or inhibiting co-medications. (Supported by Virginia Commonwealth University.) (1) [(A) Percent reduction of the number of seizures; (B) Percentage of seizure-free patients (ITT)]

Pharmacokinetics of Triclopyr (3,5,6-Trichloro-2-pyridinyloxyacetic Acid) in the Beagle Dog and Rhesus Monkey: Perspective on the Reduced Capacity of Dogs to Excrete This Organic Acid Relative to the Rat, Monkey, and Human

The pharmacokinetics of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) were measured in the beagle dog and rhesus monkey and compared with the kinetics observed in rats and humans. In addition, studies were conducted in anesthetized dogs to better understand the mechanism by which [14C]triclopyr is eliminated in this species. Triclopyr was dissolved in distilled water, and administered as a single oral dose of 0.5, 5, or 20 mg/kg to three male dogs. A single male rhesus monkey was given an intravenous dose of 30 mg [14C]triclopyr/kg body wt on two occasions separated by 10 days. Anesthetized male dogs, were implanted with venous, arterial, and urethral catheters and given increasing amounts of triclopyr to produce plasma triclopyr levels ranging from 0.3 to 27 μg eq/mL. In the monkey, triclopyr was rapidly eliminated from the plasma (t12 = 6.3 hr) with >95% of the urinary14C activity excreted within 24 hr postdosing. In the dog, orally administered triclopyr was rapidly and effectively absorbed at every dose level with virtually all of it excreted in the urine by 72 hr postdosing. However, the kinetics were slightly nonlinear, and the fraction of the dose excreted in the urine decreased with increasing dose. Several nonlinear processes may collectively contribute to the modest nonlinear pharmacokinetics in the dog. Plasma protein binding of triclopyr in the dog ranged from 94 to 99%, was nonlinear, and was an important determinant in the renal clearance of triclopyr. The nonlinear plasma protein binding indicates that glomerular filtration became disproportionately more important as plasma triclopyr concentration increased. There was good evidence for a high-affinity low-capacity active-secretory process that was saturated by low plasma triclopyr concentrations. As plasma triclopyr concentrations increased, tubular reabsorption begins to exceed secretion, resulting in decreased renal clearance. The volume of distribution, normalized for body weight, was constant across all species. While clearance and half-life could be allometrically scaled to body weight for the rat, monkey, and human, the dog had a much slower clearance and longer half-life for triclopyr elimination than predicted allometrically. These data demonstrate that the pharmacokinetics of triclopyr in the dog are markedly different than in rat, monkey, and human.

Dose-dependent distribution volumes of total and unbound valproate in guinea-pigs: consequence of non-linear plasma protein binding.

The response of steady-state distribution volume (Vdss for total and Vdssu for unbound drug) of valproate (VPA) to dose-dependent plasma protein binding was studied in guinea-pigs. Various steady-state plasma concentrations of VPA were achieved by intravenous constant infusion. The concentrations of VPA in plasma (Css for total and C(uss) for unbound drug) and various tissues (CT) were determined. The Vdss and the Vdssu were estimated based upon the apparent tissue-to-plasma concentration ratio of VPA. The results showed that the plasma unbound fraction (fu) of VPA increased significantly with dose. The Vdss was significantly increased with, while the Vdssu was significantly decreased against the increasing dose. The increase in Vdss with dose indicated an increase in tissue-to-plasma concentration ratio, which may be attributed to the increase in distribution of unbound drug from plasma to tissues subsequent to non-linear plasma protein binding. The decrease in Vdssu against the increasing dose indicated a decrease in tissue-to-unbound plasma concentration ratio, which suggests that the extravascular distribution of unbound VPA might be capacity limited and the tissue binding of VPA negligible.

The effect of competitive and non‐linear plasma protein binding on the stereoselective disposition and metabolic inversion of ibuprofen in healthy subjects

The stereoselective disposition and metabolic inversion of ibuprofen were studied in 12 healthy subjects under conditions of competitive and non-linear plasma protein binding. Each subject received each of four oral treatments according to a Latin-square design: 300 mg R(-)-ibuprofen, 300 mg S(+)-ibuprofen, 300 mg R(-)(-)+300 mg S(+)-ibuprofen, and 300 mg R(-)(-)+600 mg S(+)-ibuprofen. For a given treatment, the partial clearance of S(+)-ibuprofen was greater than that of R(-)-ibuprofen for all stereoisomeric drug species. Likewise, the unbound partial clearances of S(+)-ibuprofen were greater for most stereoisomeric drug species. There was also less difference among treatment groups when partial clearances were referenced to unbound as opposed to total plasma concentrations of enantiomer. The unbound intrinsic clearance and fractional inversion of R(-)-ibuprofen were unchanged across the four treatments, and chiral inversion was systemic, averaging 69%. In conclusion, stereoselective differences exist for the partial and composite clearances of R(-)- and S(+)-ibuprofen even when corrected for differences in plasma protein binding. However, differences among treatment groups for a particular elimination pathway are largely due to ibuprofen's non-linear binding.

Relative bioavailability of two disopyramide capsules in humans based on total, unbound, and unbound enantiomer concentrations

The relative bioavailability of two 100-mg disopyramide formulations which showed almost an 8- to 10-fold difference in their dissolution rates at pH 1.2 and 6.8 was determined in eight healthy subjects using a randomized block design. Although no significant differences in relative bioavailability were observed between the two formulations when based on the total disopyramide concentration, an almost 30 per cent difference in the extent of bioavailability was observed when assessed in terms of the unbound (+/-)- and (-)-disopyramide concentration, due probably to stereoselective nonlinear plasma protein binding. This suggests that unbound enantiomer parameters would be more sensitive to differences in bioavailability between two disopyramide formulations. Therefore, the type of concentration used would be an important factor for precise evaluation of the relative bioavailability of racemic drugs.

Stereoselective, competitive, and nonlinear plasma protein binding of ibuprofen enantiomers as determinedin vivo in healthy subjects

The plasma protein binding and competitive inhibition parameters of R(-)- and S(+)-ibuprofen were determined in vivo in 12 healthy subjects. Subjects participated in a 4 x 4 Latin square design in which oral solutions of drug were administered as 300 mg R(-)-ibuprofen, 300 mg S(+)-ibuprofen, 300 mg R(-)- + 300 mg S(+)-ibuprofen, and 300 mg R(-)- + 600 mg S(+)-ibuprofen. Unlabeled ibuprofen enantiomers were quantitated using a stereospecific reversed-phase HPLC assay, and plasma protein binding experiments were performed using radiolabeled 14C-enantiomers and an ultrafiltration method at 37C. At therapeutic drug concentrations, the protein binding of each enantiomer was greater than 99%. Furthermore, the binding of ibuprofen enantiomers was stereoselective and mutually competitive, as well as nonlinear. The bound-free data were fitted to a model in which the non-linearity of plasma protein binding and competition between enantiomers for binding sites could be accommodated. There were substantial differences in the affinity of ibuprofen enantiomers for protein binding sites (RP2 = 0.358 +/- 0.185 vs. SP2 = 0.979 +/- 0.501 micrograms/ml; mean +/- SD) but no differences in their binding capacity (RP1 = 160 +/- 86 vs. SP1 = 161 +/- 63 micrograms/ml). Although statistically significant, the differences in competitive inhibition parameters were more modest (SKI = 0.661 +/- 0.363 vs. RKI = 0.436 +/- 0.210 micrograms/ml). As a result, the intrinsic binding (i.e., P1/P2) of R(-)-ibuprofen was greater than S(+)-ibuprofen, and the unbound fraction was significantly greater for S-enantiomer vs. R-enantiomer after a given dose of R-ibuprofen or racemate.

Nonlinear pharmacokinetics of intravenous sulphadimethoxine and its dosage regimen in pigs.

The time courses of the total (Ct) and unbound plasma (Cf) concentration after the i.v. injection of 20, 50 and 100 mg/kg of sulphadimethoxine (SDM) were examined in pigs. The area under the Ct-time curve per unit dose decreased dose-dependently. Vdarea and total body clearance of Ct increased dose-dependently. The concentration-dependent plasma protein binding of SDM was evident after 50 and 100 mg/kg. The time courses of Cf en Ct after 3 doses were analyzed by a one compartment open model with nonlinear plasma protein binding. The agreement between calculated curves of Cf and Ct and the observed values, and relative constancy of pharmacokinetic parameters were obtained over 3 doses. These results suggested that the nonlinear pharmacokinetics of SDM was caused by saturable plasma protein binding. The multiple i.v. dose of SDM was based on the dosage regimen using the nonlinear pharmacokinetic model (50 mg/kg, 24 hour interval, 4 days). The observed Cf was maintained in the intended range by the dosage regimen. Therefore, the dosage regimen based on the nonlinear pharmacokinetics may allow the unbound concentration after i.v. injection of SDM in pigs to be controlled.

Saturable first-pass kinetics of propranolol.

Reduced bioavailability (F) due to hepatic first-pass extraction of an oral dose (D) is a well-known pharmacokinetic phenomenon. An integrated solution for Michaelis-Menten kinetics of the first-pass effect is derived from the maximal metabolic rate (Vm), volume of distribution (Vd), first order absorption rate constant (ka), Michaelis constant (Km), and liver blood flow (Q). F = 1 - VmVd/kaD ln (1 + kaD/QKm) This equation for single dosage can also be extended to steady state kinetics after multiple dosing in which the amount of a drug present in the hepatic circulation is considered. According to the literature, the bioavailability of a single 80 mg oral dose of propranolol (F = 0.22) increases after multiple doses Fss = 0.36). Based on the first pass equations for single dosage and multiple dosing, the maximal metabolic rate (Vm = 0.043 mg l-1 h-1) corresponding to 310 mg per day and the Michaelis constant (Km = 0.10 mg/l) were calculated for propranolol. Incorporation of nonlinear plasma protein binding in this concept may explain the lack of threshold phenomenon for a single dose of less than 40 mg propranolol. Zero order absorption kinetics could explain why cumulation kinetics seem linear even at an excessive dosage of 960 mg propranolol per day. From these derivations it may be concluded that multiple dosing, increase in plasma protein binding, high absorption rate, and increased portal venous blood flow will increase bioavailability, whereas slow release formulations, fractional drug dosage, and saturable absorption kinetics will decrease bioavailability of first-pass drugs like propranolol.

The pharmacokinetics of doxifluridine and 5-fluorouracil after single intravenous infusions of doxifluridine to patients with colorectal cancer

The disposition kinetics of a new 5-fluorouracil prodrug, 5'-deoxy-5-fluorouridine (5'dFUR, doxifluridine), were investigated in six patients with colorectal carcinoma. Each patient randomly received two single intravenous doses of 5'dFUR (2 and 4 g.m-2) on separate days. Plasma concentrations of 5'dFUR fell rapidly with terminal half-lives ranging from 16.1 to 27.7 min. A disproportionate increase in the area under the curve with increasing dose was seen in most patients. Doubling the dose resulted in a 40% decrease in nonrenal clearance (0.60 to 0.37 l.min-1) but no apparent change in renal clearance (0.32 to 0.29 l.min-1) or steady-state apparent volume of distribution (19.8 to 20.4 l). The mechanism for dose-dependence of 5'dFUR appears to be primarily due to nonlinear elimination associated with nonrenal processes rather than nonlinear plasma protein or tissue binding.

Clinical Response and Total and Unbound Plasma Concentrations After Lidocaine Overdose

Plasma and unbound concentrations and hemodynamic and CNS effects were monitored in a patient after overdose of 2 g of lidocaine HCl infused during 1 h. Hypotension, decreased heart rate, tremors, and nonresponsiveness to stimuli were observed at plasma and unbound concentrations of 17.7 and 12.0 micrograms/ml. The half-lives of decline of plasma and unbound concentrations were 3.8 and 2.7 h; the difference in half-lives was due to nonlinear plasma protein binding.

Physiologically based pharmacokinetics of valproic acid in rabbits

Protein-binding parameters of valproic acid (VPA) in rabbit serum were determined. Due to the non-linear binding, the binding percentage decreased from 91 to 41% when the serum concentration of VPA rose from 10 to 1000 μg/ml. The hepatic clearance of VPA as unbound drug followed Michaelis-Menten kinetics. A physiologically based pharmacokinetics model was adopted to interpret the overall disposition of VPA in rabbits which incorporated the non-linear plasma protein binding and non-linear intrinsic hepatic clearance. The predicted plasma and tissue concentrations were found to be in good agreement with the observed concentrations. The reason why the plasma concentration versus time curves appear to be apparently parallel in spite of remarkable changes in the unbound concentration among three different doses could be explained by the association effects in which an increase in the total plasma concentration may produce a decrease in intrinsic hepatic clearance consistent with Michaelis-Menten kinetics resulting in an increase in the unbound fraction of VPA in plasma.

Fluid Shifts and Other Factors Affecting Plasma Protein Binding of Prednisolone by Equilibrium Dialysis

The effects of drug stability, radioactive tracer purity, buffer composition, protein concentration, and fluid shifts on the nonlinear plasma protein binding of prednisolone were examined by equilibrium dialysis. Prednisolone exhibits a concentration-dependent degradation; however, the limited extent of this does not affect protein binding. Impure tritiated prednisolone used as a tracer produces incorrect, low fractional binding values with the binding parameters generated for transcortin affected more than those for albumin. Isotonic sodium phosphate and Krebs original Ringer phosphate buffers yield similar fractional binding of prednisolone and identical protein binding parameters. Fractional binding of the steroid decreases with total plasma protein concentration, but the association constants remain constant over a twofold dilution of plasma proteins. Further dilution increases these parameters. A time-dependent colloidal osmotic fluid shift during dialysis causes dilution of plasma protein concentrations and diminished drug binding. Theoretical simulations show that the osmotic fluid shifts produce the largest changes in fractional binding for compounds that are bound by low-capacity proteins with low association constants (K < 106 M−1). A mathematical equation was developed to correct bound drug concentrations and fraction bound for protein dilution caused by this effect. The fluid shifts can be prevented by the addition of dextran (mol. wt. 70,000) to the dialysis buffer in a concentration of 55% of the total protein concentration. Multiple factors can diminish the nonlinear prednisolone binding as artifacts during equilibrium dialysis, but the changes are relatively modest.

Plasma protein binding of furosemide in kidney transplant patients.

The present investigation was undertaken in order to determine the in vivo plasma protein binding of furosemide in kidney transplant patients and its possible consequence on furosemide effect. Using an equilibrium dialysis technique, serial plasma samples of furosemide taken after intravenous administration were dialyzed against an equal volume of isotonic Krebs Ringer bicarbonate buffer (pH 7.4). Dialysis was performed at 37 degrees C for 5 hr, and furosemide concentrations (total as well as free) were analyzed by HPLC using fluorescence detection. It was observed that kidney transplant patients on concomitant sulfisoxazole treatment (KT+) had a significantly greater value for percent free of furosemide as compared to transplant patients not on sulfisoxazole (KT-) (4.4 +/- 0.8 for KT+ vs. 1.7 +/- 0.3% for KT-; p less than 0.01) as well as to healthy volunteers (4.4 +/- 0.8 for KT+ vs. 1.2 +/- 0.2% for controls; p less than 0.01). In addition, kidney transplant patients not on concomitant sulfisoxazole treatment had a significantly higher value for percent free of furosemide with respect to healthy volunteers (p less than 0.05). Nonlinear plasma protein binding was also observed for one patient, who had values for percent free of furosemide ranging from 1.3 to 12.9%. However, no significant correlation was found between the fraction of the dose excreted unchanged in the urine and percent free of furosemide.