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.     

Distribution of sodium valproate in normal whole blood and in blood from patients with renal or hepatic disease

Summary Sodium valproate at a concentration of 300 µmol/l in whole blood, partitioned between the red blood cell and plasma to produce a red blood cell/plasma partition ratio of 0.20. Red blood cell uptake was proportional to percent free drug in plasma and uptake was maximal when plasma was replaced by buffer, producing a red blood cell/buffer ratio of 0.87. Reduction of plasma protein binding by plasma dilution, by increasing the total sodium valproate plasma concentration, or by renal or hepatic disease in 24 patients, caused a predictable rise in red blood cell uptake of drug. The red blood cell represented a relatively small compartment for free sodium valproate in blood, however uptake of the drug into this compartment increased considerably in states of reduced plasma protein binding. Because the concentration of drug in the red blood cell reflects free drug concentration in plasma, the red blood cell may serve as an indicator of free drug changes in blood.     

Increased plasma binding and decreased blood cell binding of quinidine in blood from anuric rats

The blood cell/plasma concentration ratio of quinidine, as influenced by the plasma protein binding, was studied in normal and anuric rats by applying incubation and equilibrium dialysis techniques on blood and plasma, respectively, from normal and anuric rats. The plasma protein binding of quinidine in anuria was increased at concentrations of unbound drug of less than 1.75 X 10(-4) M and decreased above this concentration. At an assumed "therapeutic" quinidine concentration (1 X 10(-5) M), the mean concentration ratio (total quinidine in blood cells)/(total quinidine in plasma) was 1.84 in normals and 0.46 in anuria, and the mean ratio (total quinidine in blood cells)/(unbound in plasma) was 4.45 and 1.81, respectively. As the latter ratios were concentration dependent and greater than could be accounted for by pH-dependent distribution, quinidine is presumably bound in/on the blood cells. Reduced distribution ratio in anuria, even when related to unbound quinidine in plasma, also indicates changed binding in blood cells, a finding confirmed by applying the data to modified Scatchard plot. this may have implication for the use of blood cell/plasma concentration ratio as screening procedure for the altered plasma binding of quinidine in patients.     

In vitro partition of docetaxel and gemcitabine in human volunteer blood: the influence of concentration and gender

We have performed in vitro incubations of blood from male and female volunteers with gemcitabine and docetaxel alone, and in combination, at different concentration gradients in order to investigate changes in partition between red blood cells (RBCs), total plasma and the free fraction. After extraction and sample pre-treatment, a validated high-performance liquid chromatography method followed by UV detection was used to determine the concentrations of both drugs in the different blood constituents. The partition ratio [the concentration in the erythrocytes divided by the concentration in plasma (E/P)] was calculated. The partition ratio of docetaxel varied from 0.02 to 1.44 (mean 0.35), reflecting its relatively low affinity for RBCs, probably because of its high plasma protein binding (more than 98%). For gemcitabine, the partition ratio varied from 1 to 5, reflecting a high affinity for RBCs (less than 10% plasma protein bound). The partition ratios of both drugs increased significantly with higher whole-blood concentrations, favoring uptake in the erythrocytes when plasma protein binding is saturated. Combination incubations showed a complex and unexplained interaction between gender and the influence of docetaxel on the partition of gemcitabine. We conclude that the incorporation of drugs into the RBC pool may be important for transportation to tumor tissue and efficacy. In combination, one anti-cancer agent can alter the partition ratios of other anti-cancer agents.     

Blood to plasma ratio of mefloquine: Interpretation and pharmacokinetic implications

The blood to plasma ratio of the antimalarial mefloquine has been reported to be close to 1, while other reports indicate extensive accumulation in erythrocytes. This apparent contradiction has been resolved by a quantitative examination of the compensating effects of plasma protein binding of mefloquine which almost exactly matches the extent of mefloquine accumulation in erythrocytes. The observed blood to plasma ratio of about 1 arises as the result of a balance between extensive red cell uptake and extensive plasma protein binding. Some pharmacokinetic implications of the distribution of mefloquine within blood are outlined.     

Plasma protein binding and blood-free concentrations: which studies are needed to develop a drug?

INTRODUCTION: The plasma protein binding of drugs and metabolites is known to influence their pharmacokinetics and, therefore, their effects. Evaluating the extent and the linearity of protein binding is an essential piece of information that has to be generated during drug development. Blood cell partitioning has a similar relevance. AREAS COVERED: This paper summarizes the regulatory requirements and focuses particularly on two questions pertaining to the drug development process. The first of these questions asks when is it necessary to perform detailed clinical studies on protein binding while the second asks whether the in vitro studies presently performed in plasma produce biased information. EXPERT OPINION: The authors propose that clinical ex vivo protein-binding studies should be performed on highly bound compounds (a definition of highly bound is suggested as > 95%). They also propose that in vitro studies, to measure the free drug, should be performed in whole blood, rather than in plasma, particularly if binding to proteins or blood cells is nonlinear.     

Plasma protein binding of ketanserin and its distribution in blood

The in vitro plasma protein binding and distribution in blood of ketanserin ((+/-)-3-[2-[4-(4-fluorobenzoyl)-1- piperidinyl]ethyl]-2,4(1H,3H)-quinazolinedione, R 41 468), a novel serotonin S2-receptor antagonist used in hypertension, was studied in rats, dogs and humans. Plasma protein binding of ketanserin amounted to 95.1% in healthy subjects, 88.1% in dogs and 98.8% in rats. Its blood to plasma concentration ratio was 0.70 in humans, 0.78 in dogs and 0.65 in rats. Plasma protein binding of ketanserin-ol, the main plasma metabolite of ketanserin, was 81.2% in humans and its blood to plasma concentration ratio was 1.04. The plasma protein binding of both ketanserin and ketanserin-ol was highly dependent on pH. Albumin was by far the main binding protein for ketanserin in human plasma and binding was independent of the ketanserin concentration within a very wide range. Plasma protein binding of ketanserin in elderly hypertensive patients was not significantly different from that in healthy adults. In chronic renal failure patients, whether on haemodialysis or not, the free ketanserin fraction was 40% higher than in healthy subjects. High therapeutic levels of ketanserin (0.25 microgram/ml) did not influence the plasma protein binding of diphenylhydantoin, hydrochlorothiazide, imipramine, ketoconazole, propranolol or warfarin. Out of 12 drugs, only tolbutamide at therapeutic concentrations decreased significantly the plasma protein binding of ketanserin. However, the resulting 5-20% increase of the free ketanserin fraction is hardly clinically relevant.     

The disposition of theophylline in blood in chronic obstructive lung disease

In 42 subjects with chronic obstructive lung disease receiving chronic oral theophylline therapy, the venous whole blood theophylline concentration was closely related to the total plasma theophylline concentrations (r = 0.976, p less than 0.001). The blood/plasma concentration ratio was 0.85 +/- 0.13 and was not related to the haematocrit or the free fraction of theophylline in plasma. The red blood cell theophylline concentration was closely related and numerically similar to the free plasma concentration. This indicates that the free plasma concentration is the most important determinant of red blood cell concentration, and that binding of drug by red blood cells or active uptake into erythrocytes is unlikely to occur. Whole blood concentration can be used to predict plasma theophylline concentration in subjects with obstructive lung disease in situations where preparation of plasma is inconvenient. The therapeutic range for whole blood concentration is approximately 8.5-17 mg/L.     

The use of microdialysis for the determination of plasma protein binding of drugs.

Microdialysis sampling was used for the determination of the protein binding and the free therapeutic drug concentration of drugs in plasma in vitro. Several drugs with varying extent of protein binding and for which the plasma monitoring is important were studied. To mimic the in vivo situation, an artificial blood vessel was constructed and filled with spiked plasma circulating at the flow rate of human blood at 37 degrees C. The microdialysis probe (16 mm membrane length, 20000 MW cut off) was placed in the vessel and perfused with 0.9% NaCl at 5 microliters min-1. Dialysates were collected every 10 min and were analysed by reversed-phase LC with UV detection. The free concentration of the drug was calculated by correcting the concentration in the dialysate for the recovery of the probe, which was also determined in the artificial blood vessel after the experiment. The data confirm that microdialysis is a valid alternative technique for the determination of protein binding or free therapeutic plasma concentration of drugs on a comparative basis. Reference to literature values indicates that the results of the proposed method correspond reasonably well with accepted values.     

Determination of hepatic clearance with the account of drug-protein binding kinetics

Binding of drugs to plasma proteins is commonly considered in pharmacokinetics as being in an instantaneous equilibrium. Although if the timescale of dissociation of drug–protein complex becomes comparable to the time that a drug molecule spends in blood while passing through the elimination organ, the kinetics of protein binding may influence the organ clearance. This appears possible for the compounds that have large dissociation energy from protein. Typically, the dissociation of drug–protein complex is fast. However, the longest experimentally observed average bound time of drug to human albumin was as much as 11 min, whereas the time that a drug molecule spends in blood while passing through the liver is around 19 s. The equations for the calculation of hepatic clearance (Cl_h) with the account of protein binding kinetics are derived for the well‐stirred and parallel‐tube models. It turns out that for drugs with very low extraction ratio, the influence of protein binding kinetics on Cl_h is negligible; however, for drugs with high extraction ratio, it may lead to substantially smaller values (possibly by an order of magnitude) of Cl_h compared with that provided by the common calculations.     

The influence of various factors in the in vitro distribution of haloperidol in human blood.

Haloperidol is 89.6+/-0.3% bound (mean+/-s.e. mean) in human plasma under in vitro conditions and the free drug distributes rapidly between the plasma and the cellular elements of blood. The cell/plasma partition ratio was 1.12+/-0.06 (mean+/-s.e. mean). Alteration of plasma binding by dilution with buffer showed that uptake of haloperidol by the cellular elements of blood was proportional to free drug concentration. Bishydroxycoumarin (95 ot 286 mug/ml) reduced plasma binding of haloperidol and the displaced haloperidol was taken up by the cellular blood elements. The experiments indicate that the cellular compartment of blood as well as the plasma compartment may act as a sink for haloperidol and drug displacement interactions should therefore be interpreted with a knowledge of both of these compartments.     

Plasmolysis, red blood cell partitioning, and plasma protein binding of etofibrate, clofibrate, and their degradation products

Etofibrate (I), the ethylene glycol diester of clofibric and nicotinic acids, degrades almost equally through both half-esters with half-lives of approximately 10 and 1 min in fresh dog and human plasma, respectively. The nicotinate V degrades with half-lives of approximately 12 hr and 50 min in fresh dog and human plasma, respectively. Ester III and clofibrate VI degrade by saturable Michaelis-Menten kinetics in fresh human plasma, with similar maximum initial rates and respective terminal first-order half-lives of 12 and 26 min. Tetraethyl pyrophosphate at 100 micrograms/ml inhibited human plasma and red blood cell esterases permitting plasma protein binding and red blood cell partitioning studies. The red blood cell-plasma water partition coefficient was 5.4 for 0.2-80 micrograms/ml of I. Clofibrate (VI) showed a saturable erythrocyte partitioning that decreased from 7.8 (10 micrograms/ml) to 1 (50 micrograms/ml). The strong binding of I and VI to ultrafiltration membranes necessitated the determination of their plasma protein binding by the method of variable plasma concentrations of erythrocyte suspensions to give 96.6% (0.2-80 micrograms/ml) and 98.2% (13.6-108.4 micrograms/ml) binding, respectively. Methods for the determination of the parameters of saturable and nonsaturable plasma protein binding for unstable and membrane-binding drugs by the method of variable plasma concentrations in partitioning erythrocyte suspensions are presented.     

Plasma protein binding of bepridil

The binding of the calcium-channel blocking agent, bepridil HCl (Vascor), to plasma proteins was investigated using radiolabeled bepridil and equilibrium dialysis. Greater than 99.7% of added bepridil-14C was found to freshly collected human plasma. The binding was characterized by a saturable high-affinity site (KD = 32 ng/mL = 87 nM) on alpha1-acid glycoprotein (AAG) or on an AAG-human serum albumin complex and lower affinity binding sites on albumin and other plasma macromolecules. Bepridil that is not bound to plasma proteins is extensively distributed into erythrocytes as evidenced by a red blood cell to free drug distribution coefficient of 71 +/- 7. Despite this high value, the blood to plasma ratio of bepridil averaged only 0.67 in humans, indicating that most of the circulating drug is bound to plasma proteins. Bepridil protein binding was not affected by additions of nonesterified fatty acids. Free fractions of bepridil were enhanced by addition of verapamil, nifedipine, diltiazem, disopyramide, and warfarin but only at concentrations above those achieved clinically. Bepridil was also displaced by the plasticizer, tris-(2-butoxyethyl)phosphate. Plasma obtained from a small number of angina patients prior to bepridil administration showed no differences in ability to bind bepridil compared with plasma obtained from healthy subjects.     

Using pharmacokinetics to predict the effects of pregnancy and maternal-infant transfer of drugs during lactation

Knowledge of pharmacokinetics and the use of a mechanistic-based approach can improve our ability to predict the effects of pregnancy for medications when data are limited. Despite the many physiological changes that occur during pregnancy that could theoretically affect absorption, bioavailability does not appear to be altered. Decreased albumin and alpha(1)-acid glycoprotein concentrations during pregnancy will result in decreased protein binding for highly bound drugs. For drugs metabolised by the liver, this can result in misinterpretation of total plasma concentrations of low extraction ratio drugs and overdosing of high extraction ratio drugs administered by non-oral routes. Renal clearance and the activity of the CYP isozymes, CYP3A4, 2D6 and 2C9, and uridine 5'-diphosphate glucuronosyltransferase are increased during pregnancy. In contrast, CYP1A2 and 2C19 activity is decreased. The dose of a drug an infant receives during breastfeeding is dependent on the amount excreted into the breast milk, the daily volume of milk ingested and the average plasma concentration of the mother. The lipophilicity, protein binding and ionisation properties of a drug will determine how much is excreted into the breast milk. The milk to plasma concentration ratio has large inter- and intrasubject variability and is often not known. In contrast, protein binding is usually known. An extensive literature review was done to identify case reports including infant concentrations from breast-fed infants exposed to maternal drugs. For drugs that were at least 85% protein bound, measurable concentrations of drug in the infant did not occur if there was no placental exposure immediately prior to or during delivery. Knowledge of the protein binding properties of a drug can provide a quick and easy tool to estimate exposure of an infant to medication from breastfeeding.     

In vitro partition of irinotecan (CPT-11) in human volunteer blood: the influence of concentration, gender and smoking

We have performed in vitro incubations of blood from male and female volunteers, smokers and non-smokers, with irinotecan at a gradient of different concentrations in order to investigate changes of partition between red blood cells (RBCs), total plasma and the free fraction. Since irinotecan (CPT-11) is not metabolized in vitro, there is no data available on its active metabolite SN-38. After extraction and sample pre-treatment, a validated high-performance liquid chromatography method followed by fluorescence detection was used to determine the concentration of the drug in the different blood constituents. The partition ratio [the concentration in the erythrocytes divided by the concentration in plasma (E/P)] was calculated. The partition ratio of CPT-11 varied from 0.7 to 2.8, reflecting its relatively high affinity for the erythrocyte, probably because of its only moderate plasma protein binding (65%). The partition ratios increased significantly with higher whole-blood concentrations, favoring uptake in the erythrocytes when plasma protein binding is saturated. No gender difference was detected, but we found relatively more CPT-11 in the erythrocytes of non-smokers compared to smokers. The incorporation of drugs into the RBC pool may be important for transportation to tumor tissue and efficacy. Smoking can have a significant influence on drug partition in the blood.     

Effect of low doses of heparin on the plasma binding of phenytoin and prazosin in normal man

Summary The effect of low doses of heparin on the binding of phenytoin and prazosin to plasma proteins was evaluated in four normal subjects. Heparin activates the hydrolysis of triglycerides in plasma. The ensuing increase in non-esterified fatty acids (NEFA) was more marked in vitro than in vivo and increased the free fraction (FF) of phenytoin and prazosin in plasma. The higher FF caused a change in the plasma to whole blood ratio (P/B ratio) of both drugs. The changes in FF and P/B ratio after heparin were small, but could be of significance in pharmacokinetic studies.     

The Binding of Aripiprazole to Plasma Proteins in Chronic Renal Failure Patients

The binding of drugs to plasma protein is frequently altered in certain types of renal diseases. We recently reported on the effects of oxidation and uremic toxins on the binding of aripiprazole (ARP) to human serum albumin. In our continuing investigations, we examined the binding of ARP to plasma pooled from patients with chronic renal dysfunction. We examined the issue of the molecular basis for which factors affect the changes in drug binding that accompany renal failure. The study was based on the statistical relationships between ARP albumin binding and biochemical parameters such as the concentrations of oxidized albumin and uremic toxins. The binding of ARP to plasma from chronic renal patients was significantly lower than healthy volunteers. A rational relationship between the ARP binding rate and the concentration of toxins, including indoxyl sulphate (IS) and p-cresyl sulphate (PCS), was found, particularly for IS. Moreover, multiple regression analyses that involved taking other parameters such as PCS or oxidized albumin ratio to IS into account supports the above hypothesis. In conclusion, the limited data reported in this present study indicates that monitoring IS in the blood is a very important determinant in the dosage plan for the administration of site II drugs such as ARP, if the efficacy of the drug in renal disease is to be considered.     

Drug distribution as a function of binding competition. Experiments with the distribution dialysis technique

In the distribution dialysis technique each of the two dialysis chambers contains a binding system, and a drug is allowed to distribute between them. This technique was tested by using various intracellular and extracellular binder preparations over wide concentration ranges, and model drugs selected for their known binding properties. The drugs were then tested at therapeutic concentrations in standardized systems of liver homogenate (0.5 g ml-1) and whole blood (0.02 ml ml-1). The resulting intracellular/extracellular concentrations ratios were characteristic for the binding properties of the various drugs. Thus, for imipramine, a drug with strong tissue and weaker plasma binding properties, the concentration ratios were 25 for the system homogenate/buffer, 0.8 for buffer/blood, and 15 for the competitive system homogenate/blood. In experiments with homogenates from various tissues (liver, lung, kidney, intestine, brain) and blood in the standard system, the following approximate ratios were obtained: 1 for antipyrine, 2 for phenylbutazone, 14 for imipramine (but only 8 with muscle, skin and adipose tissue). These results reflect both the individual binding to intracellular and extracellular components and the tissue/blood concentration ratios in vivo. It is suggested that distribution dialysis is an in vitro method for characterizing the distribution of the drugs. It is also concluded that drug distribution is largely determined by a binding competition between tissue and blood sites.     

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)     

On the prediction of hepatic clearance using the diluted plasma in metabolic stability assay

It was suggested that in vivo hepatic clearance, CL(h), may be predicted rather accurately with the in vitro values of intrinsic clearance, CL(int), obtained using the microsomal incubation mix containing diluted plasma, and consequently calculated by the well-stirred model equation. Conceivably the improvement could be due to the direct account of plasma protein binding in the measured values of CL(int). It is shown in this article that the prediction of CL(h) done in this manner may not yield accurate results, both substantial underestimation or overestimation of the true value is possible. The procedure may be useful to reduce the overestimation of CL(h) for highly protein bound drugs, though the obtained value of CL(h) may be far off from the correctly calculated one. The accurate way of calculating CL(h), based on the value of CL(int) obtained in diluted plasma, is presented. It takes into account both the drug protein binding in diluted plasma and microsomal binding, as well as blood-plasma concentration ratio. The prediction of CL(h) by the suggested calculation using the experimental data on CL(int), measured at different plasma dilutions for several drugs, yields consistent (dilution independent) values of hepatic clearance. It does not seem possible to avoid the measurement of plasma protein binding, microsomal binding and blood-plasma concentration ratio for an accurate and consistent prediction of CL(h), even if the value of CL(int) were obtained in the pure (undiluted) plasma. In an early stage screening using plasma in the microsomal incubation mix may be beneficial for fast metabolizing drugs with relatively high protein binding. This would reduce a possible overestimation CL(h), and also lead to the increase of the half-life in the microsomal incubation, so that it could be measured more accurately.