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.     

Differences in the binding of drugs to plasma proteins from newborn and adult man. II

Summary The binding of certain drugs to isolated fractions of plasma proteins obtained from newborn and adult man has been studied by equilibrium dialysis. For thiopental, desipramine, nitrofurantoin, sulfamethoxydiazine, meticillin and salicylic acid no difference was found between binding to the albumin fraction from newborns and adults. However, for thiopental, desipramine and promethazine binding to the globulin fraction was smaller in the newborns than in adults. Addition of bilirubin to the albumin fraction decreased the binding of nitrofurantoin, sulfamethoxydiazine and meticillin. No difference in the binding of meticillin to the albumin or globulin fractions from newborns and adults was found. The binding decreased, however, if both fractions were combined. Four mechanisms to explain the difference in binding between newborns and adults are discussed: (1) Displacement of drugs by bilirubin, (2) different binding properties of cord and adult albumin, (3) different properties of the globulins and (4) interaction of albumin with globulins in the newborn.This study was partially supported by grant Ku 156/4 of the Deutsche Forschungsgemeinschaft     

Drug recovery following buccal absorption of propranolol.

1. The effect of ampicillin, cloxacillin, flucloxacillin and sulphafurazole on bilirubin on bilirubin binding by pooled human umbilical cord serum and bovine serum albumin was studied in vitro using Sephadex gel filtration. 2. Sulphafurazole displaced bilirubin from binding; both cloxacillins displaced bilirubin from pooled cord serum but not bovine serum albumin. 4. No displacement of bilirubin by the cloxacillins from pooled cord serum could be detected at therapeutic plasma concentrations of these drugs. 5. Scatchard analysis of the interactions showed that displacement of bilirubin by these drugs occurred principally at the primary, high affinity, low capacity binding site.     

Plasma Protein Binding of Diphenylhydantoin in Man

Abstract: The plasma protein binding of diphenylhydantoin (DPH) in heparinized plasma from adult volunteers, DPH-treated adult patients with epilepsy, normal and hyperbilirubinaemic newborn infants was investigated, using an ultrafiltration technique (¹⁴C-labelled DPH). At room temperature (16 μg DPH/ml plasma) the bound fraction of DPH in 15 adult volunteers and 13 normal infants (cord blood) was 92.6 ± 0.7 and 89.4 ± 1.4 %, respectively. At 37° the unbound fraction of DPH was increased by some 60 %. Similar data were obtained in adult patients. Among a series of 15, mostly acidic drugs added in vitro in concentrations which may be obtained during therapy, only salicylic acid, sulfafurazole and phenylbutazone caused marked decrease in DPH-binding. Dilution experiments showed different patterns with regard to the bound and the unbound amounts (μg/ml) of DPH, depending on whether plasma was diluted only (constant DPH-concentration) or both DPH and plasma were diluted to the same extent. In a group of 20 hyperbilirubinaemic newborn infants a correlation existed between the unbound fraction of DPH and the total concentration of plasma bilirubin. A competition between DPH and bilirubin for similar binding sites on the albumin molecule is suggested.     

The significance of plasma protein binding on the fetal/maternal distribution of drugs at steady-state

Maternal and fetal plasma differ in their concentrations of the important drug binding plasma proteins, albumin and alpha 1-acid glycoprotein, with albumin being slightly more concentrated in fetal plasma, and alpha 1-acid glycoprotein being only 37% of the maternal concentration at term. In general, these differences relate linearly to the bound to free concentration ratio of drugs associated with these proteins. Although only the free concentration is generally considered to be the pharmacologically active form, these differences can dramatically affect the total concentration and relative distribution of drugs between maternal and fetal plasma. In order to test our hypothesis that plasma protein binding is the major determinant of fetal/maternal drug distribution at steady-state, we examined whether fetal binding could be predicted from adult binding information. Data from studies of maternal plasma protein binding were used to predict fetal plasma protein binding based solely on the differences in protein concentrations. These predictions were compared with observed fetal binding data. This analysis showed a slope near unity and a high correlation (r2 = 0.900) which implies that there are no significant differences between the binding affinities of these proteins. A similar analysis performed using data on drug binding in non-pregnant adults gave an r2 or 0.971. Having established that fetal plasma proteins bind drugs similarly to their maternal counterparts, fetal/maternal plasma drug concentration ratios (F/M) were predicted for various drugs using information from literature on the drug's adult plasma protein binding, the protein to which it binds, and the fetal and maternal plasma concentrations of that binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in the binding of quinine and quinidine to plasma proteins.

1. Little is known about the comparative plasma protein binding of the antimalarial agents quinine (QN) and its isomer quinidine (QD). We have examined the in vitro binding of QN and QD to albumin, alpha 1-acid glycoprotein, normal human plasma, and maternal and foetal umbilical cord plasma. 2. QN was more avidly bound than QD, and binding of both drugs was substantially higher to alpha 1-acid glycoprotein than to albumin, indicating that alpha 1-acid glycoprotein is the more important binding protein. 3. Protein and drug concentration dependent binding was evident for both QN and QD. The unbound fraction of both drugs fell with increasing albumin (10 to 60 g l-1) and alpha 1-acid glycoprotein (0.5 to 2.0 g l-1) concentration, and there was a marked increase in unbound fraction of QN (6 to 19%) and QD (13 to 36%) in human plasma when drug concentrations were increased over the antimalarial therapeutic range (0.5 to 10 mg l-1). 4. In human volunteer plasma, the unbound fractions of QN and QD were 7.5 +/- 2.2% and 12.3 +/- 2.3% respectively, whilst the unbound fractions for both drugs were significantly higher in maternal plasma (QN = 13.0 +/- 5.4%, QD = 18.3 +/- 2.5%) and significantly higher still in foetal umbilical cord plasma (QN = 25.7 +/- 10%, QD = 35 +/- 5.3%).     

Differential protein binding of indinavir and saquinavir in matched maternal and umbilical cord plasma

AIMS: To determine whether lower umbilical cord than maternal binding of indinavir and saquinavir contributed to the low cord : maternal (C : M) total concentration ratios reported previously. METHODS: Indinavir and saquinavir unbound fraction (fu) was determined using equilibrium dialysis. Buffer solutions of human serum albumin (HSA) (20.0, 30.0, 40.0 g l(-1)) and alpha(1)-acid glycoprotein (AAG) (0.20, 0.60, 2.00 g l(-1)) were spiked with indinavir (1.00 and 8.00 mg l(-1)) or saquinavir (0.15 and 1.50 mg l(-1)). Matched maternal and umbilical cord plasma was spiked with 1.00 mg l(-1) indinavir (n = 12) or 0.15 mg l(-1) saquinavir (n = 20). Spiked protein/plasma solutions were dialyzed against isotonic phosphate buffer, at 37 degrees C. At equilibrium, indinavir and saquinavir concentrations were quantified, and the f(u) determined. RESULTS: Indinavir and saquinavir demonstrated protein concentration-dependent binding in buffer solutions of HSA and AAG. Indinavir f(u) was significantly higher in umbilical cord (0.53 +/- 0.12) compared with maternal (0.36 +/- 0.11) plasma (95% CI of the difference -0.26, -0.097). Similarly, saquinavir fu was different between umbilical cord (0.0090 +/- 0.0046) and maternal plasma (0.0066 +/- 0.0039) (95% CI of the difference -0.0032, -0.0016). The transplacental AAG concentration gradient contributed significantly to the binding differential of both drugs. CONCLUSIONS: The differential plasma binding of both drugs, which was largely the result of the transplacental AAG concentration gradient, would contribute to the low C : M total plasma concentration ratios observed previously. Unbound concentrations of indinavir and saquinavir are likely to be substantially lower in umbilical cord than maternal plasma.     

Elevated plasma glycoproteins in fetal sheep do not bind basic lipophilic drugs

We have evaluated the contribution of nonprecipitable N-acetylneuraminic acid (NANA)-containing plasma glycoproteins found in fetal sheep plasma to the binding of three basic lipophilic drugs: propranolol, lidocaine, and methadone. In adult plasma, the measurement of NANA correlates linearly with the bound fraction of these drugs since the NANA concentration almost exclusively represents the alpha 1-acid glycoprotein (AGP) present, and AGP is the predominant binding protein for these drugs. We found that NANA concentrations in fetal sheep are nearly 20 times those of the mother which, if due to AGP, would suggest an equivalent elevation in binding. Our results opposed this hypothesis since the ratios of bound-to-free drug concentrations in fetal plasma were only 20-36% of the maternal values. From this we conclude that NANA-containing glycoprotein concentrations do not appropriately predict the binding of basic lipophilic drugs in fetal plasma, in contrast to what has been found in adult plasma.     

Characterization of peripheral-type benzodiazepine recognition sites in the rat spinal cord

The binding of [3H]Ro 5-4864 to membranes prepared from spinal cord of the adult rat has been studied in vitro. At 4 degrees C, the binding of [3H]Ro 5-4864 reached equilibrium by 120 min, and was rapidly reversible (dissociation t0.5 = 21 min). The [3H]Ro 5-4864 bound with a high affinity (Kd approximately equal to 3 nM) to a single, saturable population of binding sites (Bmax = 27 pmol/g tissue wet weight). Activation of receptors for gamma-aminobutyric acid with 10 microM muscimol did not alter these binding parameters. The drugs Ro 5-4864, diazepam and flunitrazepam were potent inhibitors of this binding (Kis of 10(-9)-10(-8) M) whereas clonazepam, CL 218,872 and Ro 15-1788 were weak inhibitors (Kis greater than 10(-5) M). A comparison of the binding of [3H]Ro 5-4864 in spinal cord with that in other areas of the CNS revealed that whereas the binding affinity was similar in all regions, membranes from spinal cord contained a slightly greater number of binding sites than cerebral cortex and cerebellum, and approximately one-third of the number present in the olfactory bulb. The characteristics of the binding of [3H]Ro 5-4864 obtained in this study are consistent with this ligand binding to peripheral-type benzodiazepine recognition sites in membranes from spinal cord.     

Plasma protein binding of drugs in pregnancy

The degree of binding to plasma proteins is an important determinant of drug disposition and response. Normal human pregnancy is associated with concentration of plasma proteins, free fatty acids and possibly other endogenous substances interfering with drug binding. The possibility of an associated change in plasma binding capacity therefore needs to be taken into consideration. Experimental studies conducted mostly in vitro have shown that the plasma protein binding of many (but not all) drugs is decreased during pregnancy, particularly during the last trimester. This phenomenon should be taken into account when interpreting serum concentrations of total (free + protein-bound) drug in clinical practice. Notable examples of drugs whose unbound fraction increases during pregnancy include diazepam, valproic acid, phenytoin, phenobarbitone, salicylic acid, pethidine, lignocaine, dexamethasone, sulphafurazole and propranolol. For many drugs, important differences have been demonstrated in the degree of protein binding between maternal and cord plasma. In some cases, this may provide an explanation for the finding of marked differences in total drug concentration between maternal and fetal plasma at the time of delivery.     

Interindividual Differences in the Protein Binding of Sulfonamides: The Effect of Disease and Drugs 1,2

Abstract: The variation in the plasma protein binding of sulfonamides in healthy adults was minimal. Additional evidence for consistency in binding came from a literature survey in which it was found that the plasma protein binding of several sulfonamides in man and laboratory animals was remarkably similar as reported by investigators from different countries. Some hospitalized patients, however, do show a binding deficiency which may be due to an interaction between drugs, disease and altered plasma proteins. Several drugs were found to compete with the sulfonamide for the same binding sites on the protein (albumin) molecule. The plasma protein binding of a sulfonamide in six anephric patients was studied before and after haemodialysis. As expected all showed a deficiency in binding. Unexpectedly, however, the binding was less in the post-dialysis plasma sample in three patients. In some patients there appeared to be a qualitative and a quantitative change in the binding sites on the protein. More definitive studies are needed with plasma protein fractions from healthy and ill subjects in the presence and absence of drugs. This may lead to a better understanding of the side effects to some drugs and could result in the development of useful displacing agents.     

Comparison of histamine release in cord blood and adult blood.

Histamine release (HR) after stimulation with anti-IgE, concanavalin A (ConA) and Formyl-Met-Leu-Phe (FMLP) from 97 cord blood samples was compared to results obtained in identically treated blood samples from adults. The maximal HR obtained with anti-IgE did not differ significantly from the values obtained in adult blood, although a ten times higher concentration of anti-IgE was required for maximum release. Passive sensitization with IgE-rich plasma caused a significant increase in maximal anti-IgE-induced HR in the majority of cord blood samples, and the dose-response curve was similar to that obtained in adult blood. Challenge with ConA and FMLP caused a HR similar to that seen in adult blood, but the close correlation between anti-IgE- and concanavalin A-induced HR seen in adult blood was absent in cord blood.     

Protein binding of non-steroidal anti-inflammatory drugs in plasma and synovial fluid of arthritic patients

1 The protein binding of seven non-steroidal anti-inflammatory drugs (indomethacin, tolmetin, salicylic acid, ibuprofen, flurbiprofen, naproxen and GP53,633) and warfarin was investigated by equilibrium dialysis in simultaneous samples of synovial fluid and plasma from 12 arthritic patients. 2 The protein binding of all drugs studied except warfarin and flurbiprofen was significantly lower in synovial fluid than in plasma. 3 The decreased protein binding of these drugs is likely to explain the lower total drug concentrations found in synovial fluid in comparison to plasma. 4 The lower albumin concentration plays an important role in determination of reduced drug binding in synovial fluid compared to plasma and the fatty acid concentration in synovial fluid may also influence the protein binding of some of these drugs.     

Prediction of drug concentrations in human skim milk from plasma protein binding and acid-base characteristics.

1. Protein binding in human skim milk of a series of seven drugs with diverse plasma protein binding and acid-base characteristics, was measured by ultrafiltration. 2. A mathematical relationship between plasma and skim milk unbound fractions was established using measured values from this study along with values from the literature. 3. The relationship enables prediction of unknown protein binding values of drugs in skim milk from known plasma protein binding values. 4. Knowledge of milk protein binding enables a more accurate assessment of total milk concentrations than is available from existing theory which is limited to prediction of unbound drug.     

Protein S modulates the anticoagulant action of recombinant human activated protein C: a comparison between neonates and adults

Recombinant human-activated protein C (rhAPC, Drotrecogin alpha (activated), Xigris) has been shown to reduce organ damage and decrease mortality in severe sepsis. Since protein S (PS) serves as a potentiating cofactor of activated protein C and since PS levels are low in neonatal plasma, we hypothesized that the anticoagulant effect of rhAPC would be decreased in cord plasma compared to adult plasma. We demonstrate that the anticoagulant action of 0.3 microg ml(-1) rhAPC (5 nmol l(-1)) was decreased in cord plasma compared to adult plasma, and dose dependently increased in cord plasma in the presence of increasing activities of PS. Correspondingly, the anticoagulant action of rhAPC decreased in adult plasma in the presence of decreasing activities of PS. The low anticoagulant action of rhAPC in cord compared to adult plasma is attributable to low neonatal levels of PS, and as previously shown, to low neonatal levels of TFPI and AT. Our laboratory experiments do not allow definite conclusions for clinical situations. However, we speculate that the anticoagulant efficacy of rhAPC is impaired in neonates and in clinical situations associated with consumption and/or inhibition of PS, AT, and TFPI, such as severe sepsis.     

Binding of drugs to human muscle

Summary Binding of 22 drugs to human muscle tissue has been determined by ultrafiltration. All drugs tested were bound, the bound fraction ranging from 13% (aminophenazone) to >98% (desipramine). Both linear and nonlinear binding was observed. For chemically related substances, binding to muscle tissue correlated with plasma binding and lipid solubility. There were significant differences in binding to muscle from different individuals. With respect to pharmacokinetics of drugs, it is suggested that binding to muscle tissue may be at least as important as plasma binding.Supported by grant Ku 156/4 from the Deutsche Forschungsgemeinschaft     

Comparative pharmacokinetics of coumarin anticoagulants XV: relationship between pharmacokinetics of dicumarol and warfarin in rats.

The distribution, elimination, and anticoagulant effect of dicumarol and warfarin were determined in adult males rats following intravenous injection of single doses of these drugs in crossover experiments. The biological half-life of dicumarol ranged from 5 to 28 hr; that of warfarin ranged from 9 to 30 hr. There was a statistically significant correlation between the following pharmacokinetic characteristics of dicumarol and warfarin in individual animals: biological half-life, apparent volume of distribution, total plasma clearance, and concentration in plasma eliciting one-half the maximum anticoagulant effect (effective concentration). The mean ratio of the respective biological half-lives (warfarin/dicumarol) was 1.42, and that of the apparent volumes of distribution was 1.50. The ratio of the effective plasma concentrations (dicumarol/warfarin) was correlated negatively with the half-life of dicumarol and positively with the ratio of the half-life values (warfarin/dicumarol) in individual animals. Additional studies with serum samples from other rats showed pronounced interindividual differences in the serum protein binding of both dicumarol and warfarin and a strong correlation between the protein binding of these two drugs in serum of individual animals. The results of this study, together with the results of previous studies in this series, indicate that serum protein binding is the major determinant of interindividual differences in the pharmacokinetics of dicumarol and warfarin in rats under these experimental conditions.     

Prediction of drug distribution in vivo on the basis of in vitro binding data

For 11 drugs it was investigated whether tissue distribution in vivo can be predicted by use of binding data obtained in vitro. The selection of drugs represented a broad spectrum of physicochemical and pharmacokinetic properties thought to be important for distribution of drugs in vivo. The extent of binding to plasma and to tissue-homogenates of rabbits was determined in vitro. The drug concentrations in plasma, liver, lungs, kidneys, and skeletal muscle of rabbits were determined in vivo after i.v. administration of the drug. The tissue-plasma partition ratios measured in vivo were compared with the theoretical tissue-plasma partition ratios calculated from the in vitro binding data. For all drugs investigated the muscle-plasma partition ratio could be reasonably well predicted by the in vitro binding data. In liver, lungs, and kidneys good agreement was found between measured and predicted tissue-plasma ratio for anionic drugs; marked differences, however, were observed between measured and predicted tissue-plasma ratios of lipophilic cationic drugs. A significant correlation was found between binding of drugs to muscle tissue in vitro and the volume of distribution of the unbound drug (Vf), opening the possibility to approximate Vf from in vitro binding studies with rabbit muscle tissue.     

The protein binding of some drugs in plasma from patients with alcoholic liver disease

Summary The binding of 5 drugs to plasma proteins from patients with alcoholic liver disease has been studied by in vitro techniques and compared to that from normal subjects. The binding of quinidine is decreased in such patients. For this reason, if in patients with alcoholic liver disease treated for coincidental arrhythmias with quinidine, plasma quinidine concentrations are measured, allowance for the decreased binding must be made in interpreting the concentration measurements. Diphenylhydantoin binding was normal or near normal in most of the patients' plasma samples. The other drugs tested, fluorescein, dapsone, and triamterene, had decreased binding in the patients' plasma samples.Supported by grants (GM 18279 and RR349) from the U.S. Public Health Service and a grant from the Smith, Kline, and French Foundation.     

Disease-induced changes in the plasma binding of basic drugs

The plasma binding of basic (cationic) drugs differs from that of the more completely studied acidic drugs. Basic drugs associate with a number of plasma constituents. alpha 1-Acid glycoprotein, lipoprotein, and albumin all appear to play an important role in the binding of most of these drugs. Acidic drugs bind largely to albumin. The variation in plasma albumin is relatively narrow and is almost always in the direction of decreased concentrations. alpha 1-Acid glycoprotein and lipoproteins show large fluctuations due both to physiological and pathological conditions. Decreases and increases in concentration have been observed. Associated with these changes in binding proteins, both decreases and increases in plasma binding of basic drugs have been recorded. Increased binding with disease appears to be virtually unique to basic drugs. The implications of these newly described disease-induced increases in plasma binding have yet to be explored. With the limited information in hand the following consequences are predicted. Increased binding will tend to decrease the volume of distribution of total (bound plus free) drug. The clearance will be unchanged or decreased depending upon the initial clearance of the drug and the avidity of the protein binding. As the half-life depends upon both clearance and volume of distribution, changes in it will be variable, depending upon changes in these two parameters. It is predicted that the area under the free drug plasma concentration-time curve will decrease with increasing binding after an intravenous dose while it will be unchanged after an oral dose. The relationship of total drug plasma concentration to free drug concentration will change with changes in binding. Thus plasma concentration monitoring of drug therapy by use of total drug concentrations will be inaccurate in situations in which large variations in binding occur. Misinterpretations of both therapeutic monitoring and pharmacokinetics studies in disease states with altered binding are likely unless these changes are appreciated.