A. Viani

Methods of determining plasma and tissue binding of drugs. Pharmacokinetic consequences.

SummaryThe available techniques for the investigation of drug binding to plasma and tissues protein are reviewed and the advantages and disadvantages of the various techniques stated. A comparison of different plasma protein binding techniques is made which shows that the size of the unbound fraction of drug may be influenced by the method used. Protein binding may be assayed by methods including equilibrium dialysis, ultrafiltration, ultracentrifugation, gel filtration, binding to albumin microspheres and circular dichroism. Tissue binding techniques can involve testing binding to isolated organs, tissue slices, homogenates and isolated subcellular particles. Details of the available methods to compute pharmacokinetic constants are given. Stereoselective binding has been investigated for a limited number of drugs and the difference in the binding of 2 enantiomers is usually modest. The measurement of the binding constants is often required to characterise the drug-protein interaction. Mathematical and graphical methods to compute the pharmacokinetic parameters are discussed. The implications of binding on the volume of distribution and clearance of drugs are examined.

Effects of Development, Aging, and Renal and Hepatic Insufficiency as well as Hemodialysis on the Plasma Concentrations of Albumin and α1-Acid Glycoprotein: Implications for Binding of Drugs

The concentrations of total proteins, albumin, alpha 1-acid glycoprotein (AGP), and nonesterified fatty acids (NEFA) were measured in the serum of 21 newborn infants and 13 children, aged 1-13 months, and in the plasma of 31 volunteers, 25 patients with renal failure, 27 patients with cirrhosis, 39 uremic patients undergoing hemodialysis, and 20 elderly subjects. The concentration of albumin in the volunteers was higher than in all other groups. The concentration of AGP in the volunteers was higher than in newborn infants but lower than in elderly subjects, patients with renal failure, and those with chronic uremia. The concentration of NEFA in volunteers was higher than in newborn infants and patients with renal failure and lower than in elderly subjects and patients with cirrhosis.

Plasma protein binding of clonazepam in hepatic and renal insufficiency and after hemodialysis.

Summary The plasma protein binding of clonazepam was investigated in healthy volunteers, cirrhotic patients, chronic uremic patients maintained on hemodialysis, and patients with reduced renal function. Each group consisted of six subjects. The unbound fraction of clonazepam (x ± SEM) was 13.9 ± 0.2% in volunteers, 17.1 ± 1.0% in cirrhotic patients, 15.6 ± 0.5% before and 12.2 ± 0.4% after hemodialysis in chronic uremic patients, and 16.0 ± 0.7% in patients with poor renal function. The figure for the healthy subjects was significantly different from that of cirrhotic patients only. Binding of clonazepam to albumin and α1-acid glycoprotein was also studied. Clonazepam bound preferentially to albumin.

Serum protein binding of furosemide in newborn infants and children.

The protein binding of furosemide was studied in the serum from 8 umbilical cords, in 51 children (aged between 2 weeks and 13.5 years) and in the plasma of 10 volunteers (aged between 28 and 42 years). The drug was added to the buffer to give a final concentration of 2 micrograms/ml. The unbound fraction of furosemide was 2.5 +/- 0.1% (cord serum) and 1.7 +/- 0.7% (adult plasma). These figures are different at a level of 0.001. The unbound fraction of furosemide reached the adult values during the 1st year of life. A correlation (level of significance greater than 0.01) was found between the unbound fraction and the age during the first 6 months of life. The furosemide binding kinetics were studied in 3 cord serum and in 3 adult plasma samples. The concentration of the drug in the buffer ranged between 1 and 16 micrograms/ml. The kinetic constants (mean +/- SEM) were: association constant (K = 10(5) M-1) 2.4 +/- 0.3 (cord serum), 2.0 +/- 0.2 (adult plasma); the number of binding sites per gram protein (n = 10(-6] was 3.2 +/- 0.5 (cord serum) and 3.9 +/- 0.7 (adult plasma). When the concentration of furosemide was increased up to 200 micrograms/ml buffer, the free fraction of the drug increased up to 4.8 +/- 0.2% (cord serum) and 2.9 +/- 0.4% (adult plasma).

Binding of diazepam, salicylic acid and digitoxin to albumin isolated from fetal and adult serum

Albumin was isolated from pooled fetal serum obtained at normal delivery at term and from pooled adult plasma. Albumin isolation was carried out by means of PEG precipitation followed by ion exchange chromatography on DEAE-Sephadex A 50 and then on SP-Sephadex C 50. The binding of diazepam (1 microM), salicylic acid (2 mM) and digitoxin (6 nM) to albumin (40 g/l) was measured by equilibrium dialysis at 37 degrees C. The unbound fraction (mean +/- SD) for fetal and adult albumin of diazepam was 1.86 +/- 0.24 and 1.82 +/- 0.15% (NS), that of digitoxin was 3.18 +/- 0.27 and 3.36 +/- 0.04% (NS) and that of salicylic acid was 11.65 +/- 0.99 and 9.47 +/- 0.75% (p less than 0.05), respectively. With both fetal and adult albumin, a single class of binding sites was observed for diazepam and digitoxin, whereas two classes of binding sites were observed for salicylic acid. The number of binding sites (n, moles of drug per mole of albumin) for fetal and adult albumin was 0.83 and 1.02 for diazepam and 0.014 and 0.018 for digitoxin, respectively. For salicylic acid, n was 1.45 (fetal albumin) and 1.55 (adult albumin) for the higher affinity site, and 3.06 (fetal albumin) and 3.27 (adult albumin) for the lower affinity site. The association constant (Ka, M-1) for diazepam was 1.36 x 10(5) (fetal albumin) and 1.00 x 10(5) (adult albumin) and that for digitoxin was 4.12 x 10(6) (fetal albumin) and 2.7 x 10(6) (adult albumin). For salicylic acid, Ka was 38.4 x 10(3) (fetal albumin) and 35.8 x 10(3) (adult albumin) for the higher affinity site, and 2.7 x 10(3) (fetal albumin) and 4.3 x 10(3) (adult albumin) for the lower affinity site. This work shows that fetal and adult albumin have similar binding properties and corroborates our previous findings with furosemide.

Plasma protein binding of alpidem in healthy volunteers, in neonates and in liver or renal insufficiency

SummaryThe binding of alpidem, a new anxiolytic drug, has been studied in plasma from 6 healthy subjects, 12 patients with renal failure, 12 patients with liver cirrhosis and 12 chronic uraemics maintained on haemodialysis, as well as in 12 serum samples from the placental cord, to represent the situation in the newborn.The unbound fraction was 0.61% (healthy volunteers), 1.31% (newborns), 0.86% (cirrhotic patients), 0.72 (patients with renal failure), 0.70% (before haemodialysis) and 0.79% (after haemodialysis). Binding in the volunteers was significantly different from that in neonates and cirrhotics only.Alpidem became bound to isolated albumin (45 g·l−1) and alpha1-acid glycoprotein (0.75 g·l−1) to 97.2% and 97.1%, respectively. The bound fraction of the drug in a mixture of two proteins was 99.1%.For alpidem, it appears that alpha1-acid glycoprotein may balance the effect of any decrease in the albumin concentration.

Plasma protein binding of furosemide in the elderly

SummaryThe protein binding of furosemide was investigated in plasma from 22 old and 11 young subjects by equilibrium dialysis. The unbound fraction of furosemide was 3.16% in plasma from the elderly and 1.71% in plasma from the young. A significant correlation was found between the unbound fraction of furosemide and the plasma concentration of albumin. The average number of binding sites was 3.8 (elderly) and 2.7 (young) 10−6 mol/g albumin. The average association constant (K) was 4.3 (elderly) and 4.2 (young) 105 M−1. By increasing the concentration of furosemide up to 200 µg/ml buffer the unbound fraction of the drug rose to 5.2% (elderly) and 3.5% (young).