Binding of amobarbital,pentobarbital and diphenylhydantoin to blood cells and plasma proteins in healthy volunteers and uraemic patients

Summary By equilibrium dialysis of human plasma it has been shown that the binding of pentobarbital and diphenylhydantoin to plasma proteins is decreased in uraemic patients (46 and 74 per cent bound, respectively) compared to healthy volunteers (61 and 88 per cent bound). The degree of binding of pentobarbital was significantly correlated with that of diphenylhydantoin and amobarbital, which suggests similarity of their binding sites. Appreciable proportions of the drugs were found in blood cells both in healthy and uraemic subjects. As expected, the distribution of drugs in whole blood was different in the uraemics from healthy subjects, because of the decreased plasma protein binding and the lowered red cell count in uraemia. Analysis of the data showed that the ratio between the concentrations in blood cells and plasma water in uraemic patients was not significantly different from that in healthy subjects.     

Age differences in drug binding by plasma proteins: Studies on human foetuses,neonates and adults

Summary The binding to human plasma proteins of ampicillin, -azidobenzylpenicillin, benzylpenicillin, phenobarbital and diphenylhydantoin was studied by equilibrium dialysis of labelled compounds. Human foetal and neonatal plasma had very low binding capacities for all five drugs as compared with plasma from adults. Hyperbilirubinemia in the neonatal period further decreased the binding capacity. The age dependence of drug binding activity should be taken into account when analysing pharmacological effects in foetuses and newborn children.     

Pharmacokinetics of meropenem in subjects with renal insufficiency

Summary The pharmacokinetics of IV meropenem (500 mg over 30 min) has been studied in 6 healthy volunteers and 26 patients with various degrees of renal impairment. Blood samples were taken at different times over 24 h in healthy subjects and 36 to 48 h in uraemic patients, and four or five urine samples were collected over 24 or 48 h. Meropenem concentrations in plasma and urine were measured by a microbiological assay.The mean peak plasma concentration of meropenem ranged from 28 to 40 g·ml^–1 and was not affected by the degree of renal impairment. The terminal half-life of meropenem was approximately 1 h in subjects with normal kidney function and it was proportionately increased as renal function decreased. A significant linear relationship between total body clearance and creatinine clearance as well as between renal clearance and creatinine clearance was observed. The mean apparent volume of distribution at steady state was not significantly altered in uraemic patients. The mean cumulative urinary recovery of meropenem in healthy volunteers was 77% of the administered dose and it was significantly decreased in patients with renal impairment. Haemodialysis shortened the elimination half-life, from 9.7 h during the predialysis period to 1.4 h during the dialysis period. The dose of meropenem should be reduced in relation to the decrease in creatinine clearance.     

In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique

The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients.     

Plasma protein binding of frusemide in renal failure rabbits: investigation of endogenous protein binding inhibitors

The reduction mechanism of frusemide-protein binding in the plasma of renal failure was investigated. The drug-albumin binding was inhibited by the low molecular-weight fraction obtained from acute renal failure rabbits, suggesting the presence of the inhibitors in the plasma. Further, this fraction was divided into six subfractions by Bio-Gel P-2. Fractions II and V2 showed significant inhibition of the protein binding of frusemide. Among uraemic toxins, four indole derivatives markedly inhibited the protein binding. Analysis by hplc confirmed that the concentration of indican was markedly increased in acute renal failure rabbit plasma. It is suggested that this compound could be one of the major inducers of the protein binding defect.     

Comparison of the serum protein binding of maprotiline and phenytoin in uraemic patients on haemodialysis

Summary The serum protein binding of maprotiline and phenytoin has been compared in a group of 22 uraemic patients receiving haemodialysis. Determination of protein binding was carried out in vitro using equilibrium dialysis at 37°C and¹⁴C-labelled drug. The mean percentage unbound maprotiline found in patients (10.0%, SD 2.5) was not significantly different from that obtained in healthy volunteers (mean 10.5%, SD 1.0). However, there was a significantly increased variability in binding in patients compared with healthy subjects. The mean percentage unbound phenytoin in the same patients (22.2%, SD 3.3) was significantly greater than that obtained in healthy control subjects (12.5%, SD 0.6). Although there was no correlation between maprotiline and phenytoin binding and serum concentrations of ₁-acid glycoprotein, there was a significant correlation between percentage unbound maprotiline and serum albumin concentrations. The findings indicate that the binding of this tricyclic antidepressant is essentially normal in uraemia, although there may be increased interindividual variability in the free fraction of drug.     

Plasma protein binding of risperidone and its distribution in blood

The plasma protein binding of the new antipsychotic risperidone and of its active metabolite 9-hydroxy-risperidone was studied in vitro by equilibrium dialysis. Risperidone was 90.0% bound in human plasma, 88.2% in rat plasma and 91.7% in dog plasma. The protein binding of 9-hydroxy-risperidone was lower and averaged 77.4% in human plasma, 74.7% in rat plasma and 79.7% in dog plasma. In human plasma, the protein binding of risperidone was independent of the drug concentration up to 200 ng/ml. The binding of risperidone increased at higher pH values. Risperidone was bound to both albumin and ₁-acid glycoprotein. The plasma protein binding of risperidone and 9-hydroxy-risperidone in the elderly was not significantly different from that in young subjects. Plasma protein binding differences between patients with hepatic or renal impairment and healthy subjects were either not significant or rather small. The blood to plasma concentration ratio of risperidone averaged 0.67 in man, 0.51 in dogs and 0.78 in rats. Displacement interactions of risperidone and 9-hydroxy-risperidone with other drugs were minimal.     

Characteristics of plasma protein binding of tacrine hydrochloride: a new drug for Alzheimer's disease

The aim of this study was to characterise the plasma protein binding of tacrine hydrochloride (THA) in vitro. Binding was assessed in the plasma of 11 healthy individuals aged 20 to 27 years using ultrafiltration followed by HPLC assay.At THA concentrations from 10 to 100 ng/ml protein binding ranged from 78.6 to 71.0%. Binding to commercially available human albumin ranged from 41.7 to 38.3% and to human ₁-acid glycoprotein from 23.1 to 12.4% over the THA concentrations from 25 to 100 ng/ml.THA binding and total plasma protein, plasma albumin and ₁-acid glycoprotein were measured in healthy young subjects (n=13), healthy elderly individuals (n=12) and patients hospitalised with acute illnesses (n=8). There were significant differences between the groups in total plasma protein, plasma albumin and in ₁-acid glycoprotein but no differences in the protein binding of THA which remained constant at about 75%. There was no correlation between THA binding and any plasma protein concentration.The THA binding was not high enough to be of major significance clinically or to reduce the validity of total plasma THA measurement in therapeutic monitoring.     

A comparison of the binding of drugs to adult and cord plasma

Summary The binding of diphenylhydantoin, imipramine, diazoxide, cephalothin and cephaloglycin to adult and cord plasma is compared. The free (unbound) fraction of diphenylhydantoin, imipramine and diazoxide in cord plasma is greater than in adult plasma. Although total protein concentration of cord plasma is significantly lower than in adult plasma, this does not satisfactorily explain the lesser binding of these drugs to cord plasma.Supported in part by NIGMS Grants 14270 and 1543. Presented in part at the Society for Pediatric Research, Atlantic City, N. J., April 1971.     

Distribution of pentobarbital and diphenylhydantoin between plasma and cells in blood: Effect of salicylic acid,temperature and total drug concentration

Summary The binding of pentobarbital, diphenylhydantoin and salicylic acid to cells in blood was found to be independent of total drug concentration within therapeutic levels. Salicylic acid displaced pentobarbital and diphenylhydantoin from plasma protein binding sites, but high levels of salicylic acid had no effect on the distribution of the other two drugs to washed blood cells. Thus, in whole blood the presence of salicylic acid decreased the fraction of pentobarbital or diphenylhydantoin bound to plasma protiens and increased the fraction of the drug in plasma water and in blood cells. Diphenylhydantoin was shown not to be bound irreversibly to blood cells and equilibration in between the inside and outside of the cells was found to be rapid (within 5 min), even at high concentrations. Binding to washed blood cells was the same at 37°C and 25°C, in contrast to plasma protein binding. It is pointed out that these effects may cause certain analytical errors, resulting in changes in plasma concentration if plasma is separated at a low temperature.     

Plasma protein binding of salicylic acid, phenytoin, chlorpromazine, propranolol and pethidine using equilibrium dialysis and ultracentrifugation

The in vitro plasma protein binding of phenytoin (diphenylhydantoin), salicylic acid, propranolol, pethidine (meperidine) and chlorpromazine was measured using an air-driven, bench-top ultracentrifuge and the results were compared with those obtained by equilibrium dialysis. For all drugs studied, except chlorpromazine, a significant correlation was found between the plasma binding results obtained by the two methods. However, only in the case of propranolol the actual binding values obtained by the two techniques were very similar. In the case of phenytoin, salicylic acid and pethidine, ultracentrifugation gave higher plasma binding values than equilibrium dialysis. Binding values obtained by the two methods for chlorpromazine were not correlated at all. This may be explained by binding of chlorpromazine to the very low density lipoprotein fraction which floats after ultracentrifugation. The described ultracentrifugation binding technique may be useful to rapidly measure the plasma binding of certain drugs in microsamples.     

Disposition of valproic acid in patients with liver disease

Summary The disposition of valproic acid (di-n-propylacetate; VA) has been studied after a single oral dose of a solution of 450 mg in 7 patients with alcoholic cirrhosis and in 4 patients recovering from acute hepatitis. The diagnosis was based on biochemical function tests and histological findings. The pharmacokinetic parameters were compared with those reported for healthy volunteers. VA in therapeutic concentration (80 µg/ml) in plasma was less bound to plasma proteins in patients with alcoholic cirrhosis (70.7±11.3%) and in patients recovering from acute hepatitis (78.1±14.1%) than in controls (88.7±5.2%). The reduced binding affected the blood/plasma concentration ratio and the apparent distribution volume Vd₍₎; the latter was increased from the normal value of 0.14±0.05 l/kg to 0.22±0.09 (p<0.05) in alcoholic cirrhotics, and to 0.20±0.07 (p=0.056) in patients recovering from acute hepatitis. The half-life of elimination T_{1/2 ()} (controls=12.2±3.7 h) was significantly (p<0.05) prolonged in cirrhotics (18.9±5.1 h) and in patients recovering from acute hepatitis (17.0±3.7 h). The plasma of total drug was not impaired, which can best be explained by the lower plasma protein binding, which might have increased the of this drug which shows restricted clearance. In addition, the plasma of free drug was significantly (p<0.02) reduced in alcoholic cirrhotics. During a two day urine collection no measurable amount of unchanged VA was recovered. There was considerable excretion of VA-conjugates, which could be hydrolyzed either by HCl or by -glucuronidase/arylsulphatase (4–23% of the dose). These percentages were in the same range as in normals (26.7±16.1%). The study indicates that elimination of VA is slightly impaired in patients with dysfunction of the liver.Supported by the Robert Bosch Foundation, Stuttgart, Federal Republic of Germany     

Studies on the Excretion of Diazepam and Nordazepam into Milk for the Prediction of Milk-to-Plasma Drug Concentration Ratios

The influence of varying protein and fat content in milk of New Zealand White rabbits on the milk-to-plasma drug concentration (M/ P) ratio of diazepam and its metabolite nordazepam following administration of diazepam was studied. At various time points after littering, a bolus dose (1.5 mg/kg) followed by a 26-hr infusion (1.8 mg/h) of diazepam was administered to freely moving rabbits via a jugular vein catheter. Milk and blood samples were collected to allow characterization of milk composition and quantitative determination of diazepam and nordazepam in milk and plasma. At steady state diazepam showed M/P ratios between 3.7 and 9.5, whereas nordazepam showed ratios between 2.1 and 4.3, respectively. The relative importance of milk protein binding and milk-fat partitioning for the excretion of a drug into milk depended on the drugs affinity to milk fat. A stepwise multiple regression analysis suggested that observed M/P ratios of diazepam could be explained by considering the fat content of milk alone. Nordazepam with a lower solubility in milk fat showed M/P ratios which could be best explained by considering protein and fat concentrations together. Using the data from the infusion studies, two recently published diffusional models to predict M/P ratios were evaluated. Neither model could accurately predict the M/P ratios of diazepam and nordazepam observed in rabbits. However, after extending the model described by Atkinson and Begg to take the actually measured partitioning between skim milk and milk fat into account, a great improvement in the predictive power for observed M/P ratios occurred. Therefore, to estimate the potential for a drug to accumulate in milk using the developed relationship, the following parameters should be measured: the creamatocrit, the skim-to-whole milk drug concentration ratio, and the free, nonionized fractions of a drug in plasma and in milk.     

Diazepam plasma binding in the perinatal period: Influence of nonesterified fatty acids

Summary The plasma binding of diazepam was determined serially in 24 women undergoing either elective induction of labour (vaginal or emergency caesarean delivery) or elective caesarean section at term and in 5 nonpregnant women requiring abdominal surgery. In the majority of pregnant patients, a marked increase in diazepam percentage free was observed during labour or prior to caesarean section, reaching a maximum, 1.6 to 3.2 fold increase at delivery or within 4 h postpartum; by the fifth day postpartum, diazepam percentage free was lower than on admission to hospital. In contrast, little change in diazepam percentage free was observed during the perisurgical period in nonpregnant patients. In parturient and surgical patients, the time courses of diazepam percentage free and plasma nonesterified fatty acid (NEFA) concentration were parallel. Bivariate regression analyses of pooled data demonstrated a strong correlation (r=0.642, p=<0.01) between diazepam percentage free and corresponding NEFA concentration and a weaker correlation between diazepam percentage free and both albumin (r=–0.319, p<0.02) or total protein (r=–0.438, p<0.01). From multiple linear regression it was demonstrated that 54% of the variability in diazepam percentage free could be attributed to plasma NEFA and albumin concentrations. NEFA displacement of plasma bound diazepam was substantiated using crystalline human serum albumin. An approximate 65% increase in plasma ₁acid glycoprotein levels was observed posttrauma in both parturient and surgical patients but was unrelated to diazepam binding events. A relationship between diazepam plasma binding changes and concurrently altered disposition of diazepam during parturition is postulated.     

The influence of blood collection technique on serum and plasma protein binding of disopyramide

Summary Serum and plasma disopyramide (D) protein binding was compared after blood was collected from four normal subjects in various Vacutainer^® tubes. The fraction of disopyramide bound to proteins in control serum and plasma was drug concentration dependent and correlated well with the capacity factor (N) associated with a high affinity protein binding site. D free fraction increased 60% at a post-equilibrium concentration of 2 µg/ml in plasma following exposure of blood to green-top Vacutainer^® stoppers due to a 60% reduction in the affinity constant associated with the high affinity protein binding site. Heparin and EDTA had no effect on the plasma protein binding of D. These results suggest a competitive inhibition of disopyramide binding to ₁-acid-glycoprotein following contact of blood with rubber Vacutainer^® stoppers.Sponsored by Grant GM-28424-01 from the National Institutes of General Medical Sciences, National Institutes of Health     

Protein binding of 2-chloro 2′-deoxyadenosine (cladribine) in healthy subjects and in patients with leukaemia

The plasma protein binding of 2-chloro-2-deoxyadenosine (CdA) at 37°;C was studied by ultrafiltration in 5 healthy volunteers, in 11 patients with haematological malignancies and in purified protein preparations. In the patients, the binding of CdA to plasma proteins was 25.0% and in healthy subjects it was 21.1%. In a solution of human serum albumin (40 g·1^–1), 24.3% CdA was bound, but less than 5% was bound in a solution of ₁-acid-glycoprotein (0.7 g·1^–1). No dependence of binding on the concentration of CdA was found within a range 25–1000 nmol·1^–1.In conclusion, due to its limited binding to plasma proteins, any change in the binding of CdA is unlikely to have a major influence on its pharmacological effect.     

Pharmacokinetics of pentachlorophenol in man

Pentachlorophenol (PCP) was given orally to three volunteers at single doses of 3.9, 4.5, 9, and 18.8 mg. Daily urinary excretion of PCP and PCP conjugated to glucuronic acid was monitored using gas chromatography with electron capture detection (GC/ECD). Based on first-order elimination kinetics an elimination half-life of 20 days was derived.To eliminate interference by the uncontrolled absorption of PCP from the environment 0.98 mg ¹³C-PCP was taken by one of the volunteers. PCP levels in urine and plasma were determined using mass spectrometry (GC/ MS) with negative chemical ionization. An elimination half-life of 17 days was found in both urine and blood. The collected data were used to calculate the clearance of PCP: a value of 0.07 ml/min was found. The long elimination half-life of PCP is explained by the low urinary clearance due to the high plasma protein binding (>96%) and the tubular reabsorption. The pH-dependency of the elimination of PCP was investigated, and a distinct increase in the daily excretion was observed following alkalinization by oral administration of sodium bicarbonate.In order to elucidate the role of the enterohepatic circulation as a possible pool for PCP in humans, the bile of cholelithiasis patients with postoperative T-drainage was investigated for PCP and compared with the corresponding urine and plasma levels, but no accumulation of PCP in the enterohepatic circulation could be observed.The daily elimination and plasma levels of PCP in a group of individuals without a specific exposition were found to range from 10 to 48 g/day and 19 to 36 g/l, respectively.     

Pharmacokinetic study of the new sulfamethopyrazine-trimethoprim combination (kelfiprim) in renal insufficiency

Summary The combination of trimethoprim (TMP) and sulfamethopyrazine (SMP) has been successfully used to treat chronic urinary tract infections. Since parenchymal involvement associated with renal insufficiency of varying degree is not infrequent in these patients, it was considered important to study the pharmacokinetics of TMP and SMP in a fixed dose combination. Four groups of patients were studied: 1) 4 patients with endogenous creatinine clearance (CL_cR) between 80 and 40 ml/min; 2) 3 patients with CL_cR between 40 and 10 ml/min; 3) 3 patients on chronic peritoneal dialysis (CAPD); and 4) 3 patients on haemodialysis. A single oral dose of 250 mg TMP and 200 mg SMP was given to each patient. Multiple samples were collected over 9 days and the following pharmacokinetic parameters were calculated: total area under the plasma level curve, slow disposition rate constant and the corresponding t_1/2, plasma clearance and the apparent volume of distribution. The results show that the two moieties of the TMP-SMP combination behaved differently in uraemic patients as fas as elimination rate was concerned. TMP was eliminated more slowly both in patients with diminished renal function and in those subjected to haemo- or peritoneal dialysis. The reduction in the rate of elimination of TMP was significantly correlated with the degree of renal impairment. The elimination of SMP, however, was not significantly affected by the reduced renal function; indeed a tendency to increase was noted, at least in dialyzed patients. However, as in patients with mild renal insufficiency (CL_cR>40 ml/min) no substantial change in plasma clearance rate need be expected, the TMP-SMP combination could be given to them in the same dose schedule as in people with normal renal function.     

The effect of ageing on plasma albumin and plasma protein binding of diazepam, salicylic acid and digitoxin in healthy subjects and patients with renal impairment.

1. Plasma albumin concentration was measured in 118 healthy subjects (aged between 18 and 87 years), in 95 renal patients with creatinine clearances between 15 and 50 ml min-1 (aged between 14 and 79 years) and in 101 uraemic patients maintained on chronic haemodialysis (aged between 27 and 83 years). 2. There was a significant (P less than 0.001) negative correlation between albumin concentration and age in healthy subjects, but no correlation in patients with low creatinine clearance or in uraemic patients. 3. The ex vivo plasma binding of diazepam (1 microM), salicylic acid (2 mM) and digitoxin (37 nM) was studied in groups of age-selected young and aged healthy subjects in patients with low creatinine clearance and in patients with uraemia. The unbound fractions of diazepam and salicylic acid were about double in old compared with young healthy subjects whereas they were similar in young and old patients with lowered creatinine clearance. In uraemic patients, ageing did not affect the binding of salicylic acid whereas the unbound fraction of diazepam was slightly but significantly greater in elderly subjects. The unbound fraction of digitoxin was independent of age in both healthy subjects and in those with renal disease. 4. Decreased plasma binding of diazepam and salicylic acid was partially corrected by extensive dialysis of plasma. The lower plasma binding of diazepam and salicylic acid associated with ageing may be ascribed to the effects of endogenous displacers and to hypoalbuminaemia. The influence of these two factors appears to be drug-dependent.     

Penbutolol: Pharmacokinetics,effect on exercise tachycardia,and in vitro inhibition of radioligand binding

Summary The pharmacokinetics of penbutolol 40 mg, its reduction in exercise-induced tachycardia, and the in vitro inhibition of radioligand binding to beta-adrenoceptors by plasma have been investigated in 7 healthy volunteers.The peak penbutolol concentration of 285 ng/ml was observed 1.2 h after administration, and the maximum of 4-OH-penbutolol of 4.76 ng/ml was found after 1.64 h. Penbutolol was detected for up to 48 h, and 4-OH-penbutolol dropped below the limit of detection after about 10 h. The terminal plasma concentration of penbutolol declined with an average half-life of 19 h.The maximum reduction in exercise-induced tachycardia was 33 beats/min 2.6 h after taking penbutolol. There was still a significant reduction of about 7 beats/min after 48 h. This effect could be adequately explained by the concentration-time course of penbutolol in combination with Clark's model of the concentration-effect relationship.Antagonist activity in plasma caused 91% inhibition of radioligand binding in vitro to beta₂-adrenoceptors on rat reticulocyte membranes 1.6 h after intake of penbutolol. By 48 h after intake, radioligand binding was still significantly inhibited (23%). The in vitro inhibition of radioligand binding by plasma showed a linear correlation with the reduction in exercise-induced tachycardia for all phases of the workload. The time course of the reduction in heart rate was completely explained by the in vitro inhibition of radioligand binding. However, it was not possible to explain the in vitro inhibition of radioligand binding by the concentration-time course of penbutolol using a simple competition model, although both variables were based on the same sampling site. When the in vitro inhibition of radioligand binding was plotted against the penbutolol concentration at the same sampling times (with both variables transformed to multiples of the apparent inhibition constant) the discrepancy became even more apparent as time-related counterclockwise hysteresis.None of the known metabolites of penbutolol can explain the discrepancy between the penbutolol concentration and the inhibition of radioligand binding in vitro. It appears that an other active metabolite is formed, which contributes to the effect in vitro and in vivo and so can explain the observed discrepancy.Dedicated to Professor Dr. med. U. Trendelenburg, Würzburg, on the occasion of his 65th birthdaySome of the results were presented at the Xth International Congress of Pharmacology (IUPHAR), Sydney, 1987