Serum protein binding of diazepam in maternal and foetal serum during pregnancy.

1 The serum binding capacity for diazepam was significantly lower in pregnancy and there was a linear correlation with gestational age. 2 The binding of diazepam was not correlated to albumin during pregnancy. 3 In cord sera there was a significantly reduced binding capacity for diazepam with albumin levels of less than 40 g/l.     

Stereoselective protein binding of verapamil enantiomers

The binding of the (+)- and (-)-enantiomers of verapamil (V) to purified albumin (40 g/L), alpha 1-acid glycoprotein (0.55 g/L) and fresh serum has been studied over a wide range of verapamil concentrations (0.055 to 22 microM). The free fraction of the pharmacologically more potent (-)-V was always greater than that of (+)-V. Similar free fractions were observed in solutions of alpha 1-acid glycoprotein ((+)-V 0.079 +/- 0.016; (-)-V 0.142 +/- 0.020) and fresh serum ((+)-V 0.096 +/- 0.009; (-)-V 0.136 +/- 0.006), however the free fraction was higher in a solution of albumin ((+)-V 0.400 +/- 0.030; (-)-V 0.572 +/- 0.029). Saturation of verapamil binding sites was observed for alpha 1-acid glycoprotein only. Enantioselective verapamil serum binding was also noted in samples collected from five healthy volunteers following oral and intravenous verapamil administration. The free fraction of the individual isomers in vitro when added to predose serum as the pseudoracemic drug ((+)-V 0.06 +/- 0.01, (-)-V 0.12 +/- 0.02) was similar to that observed for the enantiomers when studied separately in vitro, indicating that the binding of each enantiomer is independent of the other optical isomer. The free fraction ex vivo after intravenous therapy ((+)-V 0.06 +/- 0.01, (-)-V 0.12 +/- 0.02) was similar to that observed in vitro in that subjects pre-dose serum. The free fraction of both enantiomers, however, was higher after oral drug therapy ((+)-V 0.13 +/- 0.02, (-)-V 0.23 +/- 0.03). The lower binding noted may be a result of competition for serum binding sites by verapamil metabolites, which attain higher concentrations following oral dosing.     

Protein binding of warfarin enantiomers in serum of humans and rats

The protein binding of racemic ¹⁴ C-warfarin and ³ H-S(-)-warfarin was determined in individual undiluted serum samples from 31 human subjects and 11 rats. The free fraction value of the R(+) enantiomer was determined indirectly from the free fraction values of the S(–) enantiomer and racemic warfarin, a method which was verified by direct determination of the free fraction of R(+)-warfarin with the unlabeled compound at higher concentrations. The ratio (mean± sd)of free fraction values, R(+):S(–), was 1.32±0.256in man and 1.56±0.351 in the rat;the difference between species is statistically significant (p<0.025).This ratio was independent of the free fraction value of either enantiomer even though there were pronounced intersubject differences in serum protein binding of the anticoagulant. S(–)-Warfarin is eliminated more rapidly than the R(+) enantiomer in man, while the opposite occurs in rats. The results of this study rule out a species difference in serum protein binding as a factor contributing to the difference in elimination kinetics but help to explain the strong correlation in the elimination kinetics of the two enantiomers in individual rats.Supported by Grant GM 20852 from the National Institute of General Medical Sciences of the National Institutes of Health.This is paper XXVI in the series Comparative Pharmacokinetics of Coumarin Anticoagulants. Previous paper: J. T. Slattery, A. Yacobi, and G. Levy,J. Pharm. Sci. (in press).     

Influence of metabolites on protein binding of verapamil enantiomers.

This study investigated the effect of verapamil metabolites on R- and S-verapamil protein binding in plasma samples collected from subjects prior to rac-verapamil dosing and following single dose and steady state rac-verapamil dosing. In vitro studies of the effects of norverapamil, D617 and D620 on R- and S-verapamil protein binding were also performed. Protein binding of R- and S-verapamil was unchanged following single and multiple doses of rac-verapamil as compared with protein binding in pre-dose samples. In vitro, norverapamil had no effect on R- and S-verapamil protein binding up to 1000 ng ml-1. Norverapamil 5000 ng ml-1 caused a 30% increase in free fraction of both R- and S-verapamil. D617 and D620 concentrations up to 5000 ng ml-1 had no effect on R- and S-verapamil protein binding. We conclude the metabolites of verapamil have no clinically significant effect on R- and S-verapamil protein binding.     

Protein binding of chloroquine enantiomers and desethylchloroquine.

The protein binding of racemic chloroquine, its enantiomers and desethylchloroquine to plasma, purified human albumin, and alpha 1-acid glycoprotein (alpha 1-AGP) was determined by equilibrium dialysis. The binding was not concentration dependent. (+)-Chloroquine bound more to plasma (66.6 +/- 1.9%) and albumin (45.9 +/- 0.8%) than (-)-chloroquine (48.5 +/- 2.4% and 35.3 +/- 0.6%, respectively). These differences were statistically significant. (-)-Chloroquine bound more to alpha 1-AGP (47.5 +/- 0.7%) than (+)-chloroquine (34.5 +/- 0.5%). The binding of desethylchloroquine to alpha 1-AGP is higher than to albumin (38.9 +/- 0.9% and 21.1 +/- 0.4%, respectively.     

Variable binding of propranolol in human serum.

Human serum proteins were fractionated by ultracentrifugation and gel filtration. Binding of propranolol was determined by equilibrium dialysis. Propranolol was distributed to lipoproteins independent of drug concentration. Two groups of propranolol binding sites were found to be present in the protein preparation containing albumin, α₁-acid glycoprotein, transferrin and prealbumin. The first binding site with a dissociation constant of 7.5 × 10^?7 was present in number equivalent to concentration of α₁-acid glycoprotein. The propranolol binding to serum samples from 21 healthy males expressed as binding ratio B/F and per cent binding ranged from 7.5 to 19.2 and 88.2 to 95.0 respectively. The binding ratio was correlated to concentration of α₁-acid glycoprotein (r = 0.85, P < 0.001), but not to concentrations of albumin and lipoproteins. The results indicate that α₁-acid glycoprotein is the main propranolol binding protein in human serum.     

Protein binding of sotalol enantiomers in young and elderly human and rat serum using ultrafiltration

The protein binding of sotalol (STL) enantiomers was evaluated using an ultrafiltration technique with serum from young (32±2 years, n=5) and elderly (73±6 years, n=5) male and female humans, and young (8 weeks, n=4) and elderly (60 weeks, n=3) male Sprague—Dawley rats. Serum samples were collected and immediately frozen at ?20°C. Within 1 week, the serum samples were thawed at room temperature, and adjusted to pH 7.4 using 0.05 M phosphate buffer, pH 5.0. Aliquots were spiked with 250 ng mL^?1 and 500 ng mL^?1 of each STL enantiomer, placed in ultrafiltration sets (Microsep, 30K molecular weight cut-off), capped, equilibrated to 37°C, and centrifuged at 1850g for 1.5h at 37°C. Aliquots of ultrafiltrate and unspun serum were analysed for STL enantiomer concentration using a stereospecific HPLC assay. In all groups, bound fraction was less than 7% for both STL enantiomers. There were no significant differences in bound fraction between groups, or between enantiomers. Adsorption of STL enantiomers to the ultrafiltration device and membrane, evaporative loss of serum samples during centrifugation, and protein concentration in each ultrafiltrate sample were all negligible. It is concluded that the binding of STL in human and rat serum at therapeutic concentrations and physiological temperature and pH is negligible and non-stereoselective.     

Nonlinear accumulation of propranolol enantiomers.

The accumulation of (+)- and (-)-propranolol was investigated in nine subjects who received 160 mg of racemic propranolol as a single dose and then once daily for 7 days. The serum concentrations of propranolol enantiomers were measured by h.p.l.c. using a novel chiral stationary phase allowing direct resolution of underivatized propranolol. The (+)-propranolol AUC increased from 412 +/- 223 ng ml-1 h after single doses (0-infinity) to 584 +/- 279 ng ml-1 h at steady-state (0-24 h) (P less than 0.05). Similarly, (-)-propranolol AUC increased from 609 +/- 304 to 777 +/- 370 ng ml-1 h (P less than 0.05). The AUC ratio (-)/(+) was 1.52 +/- 0.36 and 1.32 +/- 0.17 after single doses and steady-state, respectively (P greater than 0.05). Therefore, nonlinear accumulation occurs with both enantiomers although there is a trend for the (-)/(+) ratio to decrease at steady-state.     

The binding of bupivacaine to maternal and foetal plasma proteins

The binding of bupivacaine to maternal and foetal plasma proteins has been investigated using a three-compartment dialysis apparatus, in which maternal and foetal plasma could be examined simultaneously. Maternal protein bound approximately twice as much bupivacaine as foetal protein per gram of protein at drug concentrations in the range 0·05 to 5·0 μg/ml. Over the same range of concentrations, 92–78% of bupivacaine was bound to maternal plasma protein but only 35–31% was bound to human albumin indicating that other proteins are involved in the binding of the drug to plasma proteins.     

Stereoselective binding of propranolol enantiomers to human alpha 1-acid glycoprotein and human plasma.

The binding of propranolol enantiomers to human albumin (ALB), alpha 1-acid glycoprotein (alpha 1-AGP) and plasma was studied. (-) propranolol is more bound than (+)propranolol to alpha 1-AGP (P less than 0.001) and to plasma (P less than 0.05). In solutions containing ALB at a constant concentration (580 mumol/l) and alpha 1-AGP at increasing concentrations, the binding of both isomers increases but the stereo selectivity is evident throughout the alpha 1-AGP concentration range examined (25-100 mumol/l).     

Different binding of propranolol enantiomers to human alpha 1-acid glycoprotein

The binding of propranolol enantiomers to human alpha 1-acid glycoprotein was studied using high performance liquid chromatography in order to provide insight into binding models and to describe individual binding parameters of both enantiomers. The binding of (-)-propranolol was shown to be saturable with one major binding site (n = 0.81, k = 2.73 x 10(5)/M). The saturation process achieved its upper asymptotic value at drug/protein molar ratio of approximately 1. In the case of the opposite (+)-enantiomer the binding isotherm did not show evidence of saturation even at higher drug/protein molar ratios (up to 50). The individual binding parameters for (+)-enantiomer were n = 0.38, k = 3.4 x 10(6)/M and n'k' = 1.39 x 10(4)/M for the saturable and nonsaturable binding component, respectively. At drug/protein molar ratio 2 the circular dichroism measurements confirmed the existence of different binding models for individual propranolol enantiomers.     

The influence of gender and sex steroid hormones on the plasma binding of propranolol enantiomers.

1. Plasma binding of tritium-labelled racemic propranolol (P) was measured by equilibrium dialysis. The unbound enantiomers were separated by h.p.l.c. after chiral derivatization. The binding of (-)-P was higher than that of (+)-P. 2. Contrary to previous suggestions, a sex difference in the plasma binding of the P enantiomers (9 young women, 12 young men) was not observed. The unbound percentage of (-)-P was 9.2 +/- 1.8 (mean +/- s.d.) in women vs 9.1 +/- 1.7 in men; for (+)-P it was 10.8 +/- 1.8 vs 10.8 +/- 2.1. 3. In the nine women, the binding did not change with fluctuating plasma oestradiol concentrations during the menstrual cycle. Testosterone cypionate doubled the circulating concentrations of testosterone in eight men but had no effect on P binding. 4. Ethinyl oestradiol (50 micrograms day-1) alone or together with norethindrone (OCD) in eight of the women produced an increase in the unbound percentage of both (-)-P (11.4 +/- 2.6 vs 9.5 +/- 1.6 for control; P < 0.001) and (+)-P (13.2 +/- 2.5 vs 11.2 +/- 1.5 for control; P < 0.001). This was due to a decrease in the plasma concentrations of alpha 1-acid glycoprotein from 0.54 +/- 0.11 mg ml-1 in control to 0.37 +/- 0.08 mg ml-1 (P < 0.001) during ethinyl oestradiol treatment. 5. Enantioselectivity in the unbound fraction of P increased with increasing total binding from a (-)/(+)-ratio of 0.93 at 84% binding to a (-)/(+)-ratio of 0.78 at 94% binding (P < 0.001).     

Controlled-release formulations of propranolol and verapamil

The in vitro and clinical behavior of new controlled-release formulations of propranolol and verapamil are reviewed. In vitro dissolution studies have proved to be of little value in determining the clinical activity of these new dosage forms. There is a difference between the blood levels found with the new formulations and those of the reference products. The once-daily verapamil product was evaluated in black hypertensive patients with promising results and suggests that this dosage form of verapamil may be successful as monotherapy for treating this patient population.     

Investigation of the maternal to foetal serum concentration gradient of dexamethasone in the rat.

The basis of the maternal to foetal serum concentration gradient of dexamethasone was investigated in the rat on day 20 of gestation. The pregnant rat was assumed to behave as a two-compartment open model with one maternal and one foetal pool exchanging with each other and each to outside. Bolus injections of unlabelled dexamethasone were made into the mother and of [3H]-dexamethasone into its foetuses and serial maternal and foetal blood samples were collected a number of times. The monitoring of both forms of the drug in each sample permitted the estimation by a non-linear least square approach of two placental and two non-placental clearances. After both maternal and foetal injections of dexamethasone, its concentrations were higher in the maternal than in the foetal serum. Placental clearance of dexamethasone from the foetus to the mother was 824 +/- 40 ml kg-1 h-1 and 8.5 +/- 1 times greater than the placental clearance from the mother to foetus (103 +/- 2 ml kg-1 h-1). Foetal non-placental clearance was zero. It is suggested that a maternal to foetal serum gradient of dexamethasone is caused by its active transfer from the foetal to maternal side.     

Influence of age on serum protein binding of propranolol

Summary The extent of propranolol protein binding was determined in three different age groups of healthy drug-free caucasian males. Volunteers selected for study were 6–15 years old, 25–36 years old and 68–76 years old. Ten milliliters of blood were obtained via venipuncture and collected in glass tubes from the subjects after an overnight fast. Binding determinations were performed by equilibrium dialysis using radiolabelled propranolol. Serum albumin and ₁-acid glycoprotein concentrations were determined in all subjects by radial immunodiffusion. The results obtained showed wide intersubject variability in the binding ratio of propranolol and serum concentrations of ₁-acid glycoprotein. Mean albumin serum concentration was found to be significantly lower in the elderly group as compared to the adult and pediatric groups (p<0.02). A positive correlation was found between the binding ratio of propranolol and the serum concentration of ₁-acid glycoprotein in all the subjects (r=+0.66,p<0.005). No significant correlation was found between the binding ratio of propranolol and the serum concentration of albumin (r=–0.03,p<0.88). These data suggest that the extent of propranolol binding is influenced primarily by serum concentrations of ₁-acid glycoprotein and not by differences in age.     

Comparison of the pharmacokinetics of verapamil in the pregnant and non-pregnant rabbit: study of maternal and foetal tissue levels

1. The comparison of the pharmacokinetics of verapamil (VER) has been studied between the non-pregnant and pregnant rabbit following VER intravenous (i.v.) bolus administration. Also studied has been VER tissue distribution in the non-pregnant and pregnant rabbit and its foetuses following an i.v. infusion of VER. 2. When the pharmacokinetic variables were compared between the pregnant and non-pregnant rabbit, it was observed that t(1/2)lambda2 V1 and V(D) were significantly higher in the non-pregnant than in the pregnant rabbit. Moreover, lambda(z) was significantly lower in the non-pregnant than in the pregnant rabbit. However, AUC and CL showed no significant differences between the pregnant and non-pregnant rabbit. 3. When tissue concentrations were examined, it was found that in most of the tissues studied high concentrations of VER were found both in the pregnant and non-pregnant rabbit. Furthermore, VER concentrations in the uterus, heart, spleen and kidney were significantly higher in the non-pregnant than in the pregnant rabbit. 4. The results suggest that VER diffuses poorly through the placenta, given that VER blood concentrations were lower in blood foetuses than in maternal blood. Moreover, the concentrations of VER in the selected foetal tissues were either similar (brain and liver) or lower than those observed in maternal tissues.     

Comparison of the pharmacokinetics of verapamil in the pregnant and non-pregnant rabbit: study of maternal and foetal tissue levels

1. The comparison of the pharmacokinetics of verapamil (VER) has been studied between the non-pregnant and pregnant rabbit following VER intravenous (i.v.) bolus administration. Also studied has been VER tissue distribution in the non-pregnant and pregnant rabbit and its foetuses following an i.v. infusion of VER. 2. When the pharmacokinetic variables were compared between the pregnant and nonpregnant rabbit, it was observed that t_1/2lambda_z, V₁ and V_D were significantly higher in the non-pregnant than in the pregnant rabbit. Moreover, lambda_z was significantly lower in the nonpregnant than in the pregnant rabbit. However, AUC and CL showed no significant differences between the pregnant and non-pregnant rabbit. 3. When tissue concentrations were examined, it was found that in most of the tissues studied high concentrations of VER were found both in the pregnant and non-pregnant rabbit. Furthermore, VER concentrations in the uterus, heart, spleen and kidney were significantly higher in the non-pregnant than in the pregnant rabbit. 4. The results suggest that VER di uses poorly through the placenta, given that VER blood concentrations were lower in blood foetuses than in maternal blood. Moreover, the concentrations of VER in the selected foetal tissues were either similar (brain and liver) or lower than those observed in maternal tissues.     

Protein binding of diazepam and digitoxin in uremic and normal serum.

The protein binding of diazepam and digitoxin in serum from uremie patients has been studied by equilibrium dialysis and compared to that in normal serum. Comparisons have also been made with isolated human serum albumin (HSA) from uremie and normal individuals. Diazepam and digitoxin are bound to different sites on HSA. Their binding was impaired in the serum from the patients when compared to that in the normal serum owing to decreased affinity constants for the binding to the primary sites on albumin. In the uremic serum the number of binding sites for diazepam is increased compared with the number in normal serum and HSA. For digitoxin the number of binding sites is larger both in the normal and the patient serum than that obtained with HSA. The fact that apparently an increased number of binding sites is made use of, is probably due to the presence of substances which inhibit the binding to the primary sites. The binding of the drugs was improved after charcoal-treatment of the uremie albumin at pH 3.0.