Free drug measurements: methodology and clinical significance

Clinica Chimica Acta, 151 (1985) 193-216

193

Elsevies

CCA 03285

Critical Review

Free drug measurements: methodology and clinical significance Tai C. Kwong Depariment of Pathology and Laboratory Medicine, The University of Rochester Medical Center, 601 Efmwood Ave., Box 608, Rochester, NY I4642 (U.S.A.1 (Received

February

14th, 1985;

revision June 14th, 1985)

Key wor&: Free drug; Free drug level; Free drug concentration; Free drug measurement; Free drug rno~~tori~g

Introduction Drug molecules circulate in blood in two forms: those in association with blood components such as proteins, red cells, or platelets and those which are unbound and are dissolved in plasma water [l-3]. The extent of drug binding to plasma proteins varies widely among different types of drugs. The protein-bound fraction ranges from less than 0.1 for caffeine and ethosuximide to greater than 0.99 for highly bound drugs such as warfarin and dicumerol[4]. The effects of changes in the extent of protein binding on free drug fraction and on drug metabolism are significant only for highly bound drug (greater than 80% bound), since even a relatively small change in the degree of binding has a dramatic effect on the free fraction. Albumin is quantitatively the most important drug binding protein for many drugs, particuIarly the neutral and anionic (acidic) drugs such as warfarin, phenytoin, and valproic acid [.5]. In recent years, cationic (basic) drugs such as propranolol quinidine, and the tricyclic antidepressants have been shown to bind not only to albumin, but also to other blood proteins such as &,-acid glycoprotein (AAG) and lipoproteins f6-8). In some cases, binding to AAG and lipoproteins exceeds that to albumin. Fluctuations in plasma levels of AAG and lipoprotein fractions such as low density lipoprotein are common and point to the importance of understanding the interactions of cationic drugs with these proteins f&9,10]. Table I lists those drugs which are commonly encountered in clinical chemistry laboratories. Protein binding has an important effect on the pharmacokinetics of drugs. The impact of alterations in protein binding on drug clearance depends on the clearance characteristics of the drug. Restrictively cleared drugs are those that are removed from plasma by glomerular filtration through the kidneys or by passive uptake by the liver. Only the free fractions of such drugs are cleared by these organs. Thus, clearance of highly protein bound and restrictively cleared drugs are sensitive to

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194 TABLE Protein

I binding

of drugs

Drug Acetaminophen N-Acetyl procainamide Amikacin Amitriptyline Carhamazepine Chloramphenicol Chlordiazepoxide Chlorpromazine Desipramine Diazepam Digitoxin Digoxin Disopyramide Ethosuximide Gentamicin Imipramine Lidocaine Lithium Methotrexate Nortriptyline Phenobarbital Phenytoin Primidone Procainamide Propranolol Quinidine Salicylic acid Theophylline Tolbutamide Valproic acid Warfarin ’ h ’ d

81% at 1.7 pg/ml; 66% 95% at 14 pg/ml; 80% 34-388 by equilibrium 95% at 30 pg/ml; 75%

s Bound

Proteina

0

II

10 4

96 82 53 97 89 92 99 90 25 66-81 L’ 0 leas than 10 89-94 70 0 45 92 51 89 20-25 16 88-95 71 80-95 34-45 93 93 “ 99

h L

at 5 pg/ml. at 300 pg/ml. dialysis; 41-45% at 160 pg/ml.

ALB.AAG,LP ALB. AA<;? ALB ALB ALB, AAG. LP ALB. AAG. LP ALB ALB ALB ALB. AAG

ALB, AAG. LP ALB. AAG ALB ALB, AAG ALB ALB ALB ALB, AAG. LP ALB. AAG, LP ALB ALB ALB ALB ALB

I2 13 14 IS.16 17 IX x.19 19.20 21 22 23 24.25 26 27 2X.29 30 31 32 14.33 34 35 36.37 12 3x 39.40 41 42.43 44 45 46

by ultrafiltration

alterations in protein binding. A higher free fraction will lead to higher clearance rates, shorter elimination half-lives and a larger fluctuation in peak and trough levels. Theophylline and warfarin are two of such ‘restrictively cleared’ drugs [42,46-481. The different clearance rates of theophylline and warfarin (0.7 and 0.05 ml/min/kg, respectively) are related to their free fractions (0.62 and 0.01, respectively). Non-restrictively cleared drugs are extracted by active mechanisms in both free and bound forms by the liver or the kidneys. Clearance rates are dependent on organ blood flow and relatively independent of protein binding [49]. Lidocaine and propranolol are examples of non-restrictively cleared drugs which are highly bound

195

and which are extensively cleared by the liver in one pass (first pass clearance) [50-521. A widely held view on the pharmacological effect of protein binding is that only the free form of the drug can cross capillary and cell membranes. It is therefore assumed that the free fraction in vitro is equivalent to the pharmacologically active fraction, in vivo, and free drug levels will correlate better with drug effects and clinical conditions of the patients. It should be appreciated that the free drug hypothesis is unproven. In fact, the basic assumption that only unbound drug can cross cell membrane has been challenged in recent studies on the lipop~lic drugs lidocaine and propranolol and some hormones [53,54]. It was elegantly demonstrated that globulin-bound lidocaine and propranolol were also transported into tissues such as brain [54]. Thus, equilibrium measurement of plasma free drug, in vitro, may underestimate the amount of exchangeable plasma drug, in vivo, that is available for transport into tissue. Such measurements, however, can still reflect the levels of exchangeable drugs, in vivo, but only when transport and tissue factors are constant. Under these conditions plasma free drug levels should correlate well with clinical conditions. Despite the limited understanding of the dynamics of exchangeable plasma drug, a great deal of interest has been focused on the .clinical significance of measuring free (unbound) drug levels in plasma or serum. Much effort also has been directed to understanding pathophysiological factors, in vivo, and experimental parameters, in vitro, that influence the binding of drugs to plasma proteins. The rationale for such studies has been that measurement of free drug levels will improve the therapeutic aspects of patient care. This review will consist of three major sections. In the first section the two most frequently used techniques in drug-protein binding studies will be assessed. The theoretical limitations, the nature of the errors inherent in these techniques, and the pitfalls in inte~retation of binding data will be critically examined. Part 2 consists of a review of the literature on alterations in drug binding and on the effects of such changes on drug metabolism and pharmacokinetics behavior. In Part 3 the clinical relevance of altered drug binding in the context of the material presented in the preceding sections will be discussed. Finally, the clinical utility for free drug measurement will be addressed. Analytical techniques

Many techniques have been used to study the binding of drugs to proteins and to measure the free drug fraction. Among these are equilibrium dialysis, ultrafiltration, ultracentrifugation, and gel-filtration [55-571. In all cases, a physical separation of the free drug from the binding macromolecules is required. Equilibrium dialysis is employed most frequently, although in recent years ultrafiltration has gained considerable popularity. This review will focus on ~uilib~um dialysis and ultrafiltration because these two techniques are the most likely candidates for adoption in clinical laboratory settings.

E~u~~~bri~~~ ~i~~~~~s~~s

Aithough equilibrium dialysis is considered by many to be the preferred method for determining drug-protein binding, certain theoretical limitations and their consequences have not always been fully appreciated. A number of reviews on equilibrium dialysis are available [55,58,59]. The technique is based on the principle that unbound drug will equilibrate across a semi-permeable membrane which is permeable to small drug molecules but not to drug binding proteins. The method is performed by placing serum or plasma in a chamber separated from buffer in another chamber by the membrane. At equilibrium, the drug concentration in the buffer compartment (dialysate) is considered to represent the unbound drug concentration which will be the same as the free drug concentration in the protein compartment (retentate). Under rigorously controlled experimental conditions such as temperature. time and buffer composition, equilibrium dialysis can give reproducible results. A lack of full understanding of equilibrium dialysis, (either of its theoretical concepts or practical problems) can result in improperly calculated data. A number of papers which critically addressed these problems have appeared ]58,60-663. The most common conceptual error is the failure to appreciate that during dialysis diffusion of free drug from protein compartment into the dialysate will result in a smaller total drug concentration in the plasma and a new equilibrium [60.66]. If binding of a drug to protein is dependent on drug concentration, then the free fraction that exits at post-dialysis equilibrium is related to the lower total drug concentration inside the protein compartment at equilibrium and not to that in the initial protein sample. In other words, the bound and unbound fractions at equilibrium are different from those of the original sample. The magnitude of the difference is dependent on the volume ratio of dialysis buffer to protein sample. the affinity constant and the concentrations of drug and protein samples [60,68]. The effect of varying the ratio of dialysis buffer volume to sample volume was tested with a testosterone albumin equilibrium dialysis system, with the initial concentrations of testosterone and albumin kept constant [66]. An increase in the buffer to sample ratio from 2: 1 to 20: 1 resulted in a change of the bound/free ratio from 3.22 to 4.43, and of the percent unbound from 23.7 to 18.4. When the initial concentrations of testosterone were varied to construct a Scatchard plot, the affinity constant K, was 1.02 x lo4 for a 2: 1 buffer to sample ratio and 0.30 X 10” for a 20: 1 ratio. The K, obtained from a plot constructed from data which have been corrected for post dialysis concentration was 2.37 X 104. Thus, a significant error can be incurred if dilutional shift is not taken into consideration. In experiments to measure the extent of drug binding of an endogenous drug in plasma, an unbound labeled drug is added to the buffer compartment. A new equilibrium will be established based on the new total drug concentration which is the sum of the endogenous drug in plasma and the added labeled drug. Determination of free and bound drug will give values which are relevant to the new post-dialysis equilibrium and are not the same as those in the original sample unless the labeled drug is added at a concentration identical to the concentration of the endogenous free drug in the plasma under examination [62].

197

Other problems which should be considered in obtaining accurate results are: (a) significant Donnan effect; (b) osmotic dilution; (c) nonspecific adsorption of free drug, protein molecules or drug-protein complexes to the surface of dialysis apparatus; (d) lack of attainment of equilibrium; (e) radiochemical impurity and (f) protein leakage into dialysate. The Donnan equilibrium effect stems from unequal distribution of diffusable charged ions (such as ionized drug molecules) due to the confinement of nondiffusable charged protein molecules to one compartment by the semi-permeable membrane f67]. It is well-known that the Donnan effect will affect the determination of drug binding by equilibrium dialysis [68j, particularly for those drugs which are highly ionized and weakly bound. In principle the Donnan effect can be minimized by increasing the concentration of diffusible electrolytes or by choosing a pH close to the isoelectric point of the protein. Addition of small electrolytes (NaCl or KCl) to a final concentration of 0.15 mol/l in the system is expected to abolish the unequal distribution of diffusible ions [67]. These remedies, however, may not be applicable if drug-protein interaction is primarily electrostatic in nature, or if the binding protein precipitates at its isoelectric point. Appropriate correction of equilibrium dialysis results is possible using the Donnan ratio which can be calculated from drug ionization and distribution of non-binding ion, such as cesium ion [69]. Dilution of protein sample (retentate) due to osmotic pressure of plasma protein can lead to considerable variation in retentate volumes at final equilibrium. Accurate and precise determination of retentate volume is more difficult than for protein concentration. Thus, measurement of starting and retentate plasma protein concentrations will allow correction for dihrtion. Drug, drug-protein complex or protein may adsorb to dialysis membrane or to surfaces of the dialysis apparatus. Adsorption appears to be highest among highly ionized drugs and may be greater than 50% for some drugs as streptomycin [55]. Adsorption is dependent on the concentration of the drug. For example, as the amount of suxamethonium adsorbed to dialysis membrane increases with increasing drug concentration, the binding data could be erroneously interpreted to suggest that suxamethonium binding to protein decreases with increasing drug concentration. Blanks have to be made to eliminate the error due to adsorption. Non-specific loss of drug does not affect the calculation of free fraction if both the dialysate and retentate are assayed for drug levels [SS]. For drugs whose binding to protein varies with drug concentration, the free fraction determined after equilibrium dialysis relates to the equilibrium total drug concentration after changes due to osmotic dilution, nonspecific adsorption to membrane, and loss of free drug to the dialysate. Thus, the true free fraction of variably bound drug in the starting plasma must be calculated [60,70]. Other factors such as temperature, pH, and buffer may significantly alter the determination of drug binding to proteins, in vitro, and may account for the variation in reported values of free fractions of some drugs. In general, protein binding decreases at higher temperature; binding of theophylline was 8.9% and quinidine was 6.0% lower at 37 than at 25°C [42,71]. Dependency of protein binding on pH has been reported for a number of drugs including warfarin [72], propranolol

[73]. imipramine [74], theophylline [42.75]. quinidine and prazosin [75]. The average bound fraction of theophylline to human serum adjusted to pH 7.4 was 10.9% lower than that for the same sera without pH adjustment (pH ranged from 7.6 to 8.7) [42]. The implication of variation in binding with pH is that without careful control of pH during equilibrium dialysis, the binding data may not reflect conditions. in vivo. Buffer composition significantly influences binding values determined by equilibrium dialysis [75-771. Krebs-Ringer bicarbonate and phosphate buffers gave higher unbound fractions than isotonic phosphate buffer for quinidine 1771 and theophylline [42]. Lowering the chloride concentration in the Krehs-Ringer buffers by half decreased the free fraction of quinidine in normal serum from 22.2 to 13.5%. This is consistent with previous Findings that chloride displaces quinidine from plasma proteins (781. Ultrufiltration

Ultrafiltration is the other commonly used technique for studying drug binding to protein. A pressure gradient, which can be conveniently generated by centrifugation, forces plasma water and small molecules through a semi-permeable membrane. Ultrafiltration is an attractive alternative to equilibrium dialysis because of the ease and speed with which it can be accomplished. Speed is an important consideration in studying the binding of drugs to albumin since binding of some drugs may be decreased by fatty acids [79,80]. Fatty acid released through lipoiysis of plasma triglycerides can be significant in tong storage and in dialysis of long duration [81,82]. A major concern in using ultrafiltration has been the effect of the removal of free drug on physiologic binding equilibrium. For this reason it has been suggested that only a small fraction of the totat sample (often less than 10%) should be ultrafiltered to avoid disturbance of protein binding equilibrium [57,83,84]. This, however, makes measurements of low levels of drug in a small volume of ultrafiltrate analytically difficult. Recent reexamination of the thermodynamic aspects of ultrafiltration has indicated that ultrafiltration does not inherently alter binding equilibria [57.83,85]. Even as protein and total drug concentration increase markedly during ultrafiltration, the drug-protein molar binding ratio remains constant. Therefore, the drug concentration in the ultrafiltrate also remains constant and will be the same as the equilibrium free drug concentration in the original sample. Drug concentrations in the ultrafiltrate will also be independent or the volume of ultrafiltrate. The above theoretical prediction holds true if the association constant and the number of binding sites per protein molecule remain unchanged. The association constant may vary at very high protein concentration which may lead to protein ~onformati#nal changes or formation of aggregates. The membrane used for ultrafiltration is assumed to act as a perfect molecular sieve with no discrimination between water molecules and the larger drug molecules. A non-ideal membrane will allow higher fiitration rate for water over drug molecules. This will result in dilution of drug concentration in the uItrafiltrate which wit1 be exaggerated at higher gradient pressure. Thus, it has been observed that unbound digoxin varied from 40-75% and unbound ouabain varied from BO-97% when ultrafiltration was performed at

199

pressure ranging from 45 to 2 pound per square inch [86]. It can be expected that this molecular sieving effect will be greater for drug molecules of higher molecular weight. The error in a protein free solution is about 2% for a drug of molecular weight of 300, increasing rapidly to 13% for streptomycin (mol. wt. 581) [Ml. Ultrafiltration assays need to be vigorously validated and controlled. For each drug binding system, the effects of pH and temperature variation must be established, In general, drug binding decreases with increasing temperature, and ultrafiltration is best performed in a temperature controlled centrifuge to minimize the effect of heat generated by the centrifuge motor. Pressure gradient should be low but sufficient to generate acceptable filtration rate and is best maintained by low speed centrifugation (1000-2000 x g). Sieve effects due to polarization of protein on membrane may be minimized by centrifugation in a fixed angle rotor. Adsorptive loss to membrane has to be measured. The presence of Donnan effect in ultrafiltration is well-documented and can be corrected by the method similar to that for equilibrium dialysis [57]. Since the limitation of ultrafiltrate volume to lo-20% is arbitrary and has no theoretical basis, larger volume of ultrafiltrate can be generated to facilitate drug measurement. The volume of ultrafiltrate that can be obtained is limited by practical considerations such as the viscosity of the remaining serum and the length of time for centrifugation. Ultrafiltrate drug concentration remains the same when up to 40% of the total volume has filtered. Factors influencing drug binding, in vivo Physiologic and pathologic factors that change the extent of drug binding will result in an altered free fraction. Depending on the mechanism of drug metabolism, free drug concentration and drug clearance can also be affected. The following sections will examine these factors which affect drug binding in vivo. Ph~s~o~ogic~lfactors

There have been a limited number of studies focusing on sex-related differences in protein binding of drugs [87]. Lower protein binding in women than in men of comparable age have been observed for imipramine [88], chlordiazepoxide [89], and nitrazepam [90]. The differences are generally small but statistically significant. Reports on sex-related differences in protein binding of diazepam are conflicting [91-961. Studies on protein binding of lidocaine [93], lorazepam [97], oxazepam [98] and propranolol[96] did not reveal any differences between men and non-pregnant women. Women on oral contraceptives had significantly lower binding for chlordiazepoxide, lidocaine and diazepam than contraceptive-free females [89,93]. AAG, a major protein binder for lidocaine is as much as 35% lower in plasma concentration in women taking estrogen-containing oral contraceptives 193,991.The reduced binding of lidocaine in females on oral contraceptives has been attributed to the lower plasma AAG concentrations. Pregnancy is associated with physiological changes which have important influence on drug metabolism and disposition [lOO,~Ol]. Changes in serum levels of protein, free fatty acids, or other endogenous substances affect the extent of drug binding [102,103]. Increase in free drug fraction in patients during the last trimester

200

when compared to controls have been observed for phenytoin (l&20%) [104 1061, phenobarbital (12%) [104]. valproic acid (55’%} 11041, diazepam (44462%) [46,105-1071, lidocaine (50%) [961, salicyhc acid (80%) [105,108], sulfisoxazole [105], and propranolol (25%) [96]. Furthermore, the free fraction in pregnant women correlated negatively with serum albumin concentrations which progressively decline during pregnancy. Other pregnancy-associated factors which may be important in influencing binding capacity are elevated free fatty acid levels [109], as well as reduced binding affinity of albumin of which there is some evidence [108,110]. The effect of age on protein binding and drug disposition have been extensively reviewed [ill-1131. The free fraction of several drugs such as hdocaine [96.111], bupivacaine 11151, meperidine [116], and propranotol [96] is much higher in fetal (cord) plasma compared to maternal plasma. As binding of these drugs to AAG is substantial, the difference in maternal and fetal protein binding was probably accounted for by the two to three times greater plasma concentration of AAG in the mother than in the fetus 190.1141. For drugs which bind primarily to albumin such as diazepam [117,1 IS], valproic acid [119,120], phenobarbital and phenytoin [34], clonazepam [121] and salicylate [122], the fetal free fraction is lower. This may be because of the higher concentrations of albumin in fetal plasma and therefore higher binding [122]. Furthermore, the higher maternal concentrations of free fatty acids can competitively displace drug from albumin binding sites, making a greater fraction of drug available for placenta1 transfer. This transplacental binding gradient explains the observation that total drug levels at delivery are higher in the newborn than in the mother [117,120]. In the neonatal period, the doubling of diazepam free fraction during the first day of life has been correlated with the greatly increased free fatty acid concentrations observed shortly after birth. This. in conjunction with slow drug metabolism due to hepatic immaturity can result in unexpectedly high free drug concentration which may explain the adverse effects observed in some neonates of diazepam-treated mothers [ 118 J. Reduced protein binding in neonates also may be related to the quaIitatively different fetal albumin. Albumin in newborns differs from that of the adult [123] in having lower binding affinities for drugs [117,124,125] but higher affinity for bilirubin [124]. High levels of unconjugated bilirubin have been reported to affect the binding of diazepam 11241 and phenytoin [126,127]. In the geriatric population, decrease in drug binding to albumin has been correlated to a decline in plasma albumin concentration [128]. The decrease in albumin levels may be significantly large among poorly nourished and severely ill elderly patients [128-1301. Pathological factors

Various disease states influence the binding of drugs to plasma proteins by pathology-induced changes in the plasma concentrations of the major drug-binding proteins. For drugs which bind primarily to albumin, the literature abounds with reports of reduced drug binding in disease states associated with hypoalbumin~mia. These have included liver disease, renal failure, nephrotic syndrome, protein losing enteropathy and burns [5,131].

201

In patients with liver disease, hypoalbuminemia is primarily due to albumin loss into interstitial spaces as well as to compromised synthetic capacity for albumin. For a variety of drugs, binding to plasma proteins is reduced in patients with liver disease [132,133]. These have included phenytoin [35,134], quinidine [135], propranolol [136], the benzodiazepines [137-1391, and verapamil [140]. The lower binding of phenytoin has been associated with acute viral hepatitis [141], alcoholic liver disease [135], and liver failure [134], and have been correlated with albumin [35,135,141] and total bilirubin concentrations [142]. The association of renal failure with abnormal drug binding to albumin has long been appreciated [143,144]. Since the effect of uremia on drug binding relates to albumin, the drugs most affected are the anionic and neutral drugs such as phenytoin [22,35,134,143,144-1461, valproic acid [147-1491, phenobarbital 11501, phenylbutazone [151], salicylate [150,152], thyroxine (1531, and warfarin [151,154], and those cationic drugs such as diazepam [155] and oxazepam [156] which have significant binding to albumin. As expected, protein binding of those cationic drugs such as propranolol 11571, quinidine [157,158] and verapamil [159] which bind to AAG and lipoproteins are minimally affected by renal disease. Decrease in drug binding due to hypoalbuminemia in the nephrotic syndrome has been studied. It was observed that for each 1 g/l fall in serum albumin below 37 g/l, there was a 1% decrease in the percentage of bound phenytoin [160]. In uremic patients reduced binding has been attributed to qualitatively different albumin molecules, or to displacement of drug molecules from albumin binding sites by the organic molecules which accumulate in uremia. Evidence supporting either hypothesis has been summarized in a recent review [161]. The preponderance of the evidence, however, suggests that in uremic patients the accumulation of endogenous material which competes for albumin binding sites is probably the more important mechanism of reduced binding to albumin. As a response to tissue injury, a number of plasma proteins increase in concentrations. Among these acute phase reactants are alpha-l acid glycoproteins (AAG) and lipoproteins [10,162]. The cationic drugs such as lidocaine, propranolol, quinidine, imipramine and diltiazem bind not only to albumin, but also significantly to AAG and lipoproteins [6,9]. As AAG and lipoprotein serum levels increase following tissue injury, protein binding of cationic drug will also increase [6]. This has been reported for propranolol, lidocaine, disopyramide and i~pra~ne after acute myocardial infarction [163-l@], quinidine and propranolol after surgery [169,170], lidocaine, propranolol and methadone in cancei patients [171-1731, lidocaine in trauma [174], imipramine after severe burn injury [175], and propranolol and chlorpromazine in patients with Crohn’s disease and inflammatory arthritis 11761. Concentration-dependent

binding

At therapeutic concentrations, most drugs do not saturate the binding capacity of the binding proteins. Notable exceptions are valproic acid [17’7],salicylate [178,179] and disopyra~de [180]. Higher ~ncentrations of these drugs are associated with higher free fractions. Valproic acid free fraction jumped from 0.05 to 0.25 at plasma levels between 30 and 160 pg/ml. It was also observed that valproic acid free

fraction varied more than two-fold during a single dosing interval (177). Since valproic acid has a low extraction ratio and it restrictively cleared bv the liver, increase in free fraction after increasingly higher doses will lead to higher drug clearance and, as a result. lower steady state total drug levels. Thus. the hyperbolic level vs dose relationship for valproic acid is attributable to its concentration-dependent binding to albumin [177]. Within therapeutic range. the free fraction of salicylate increases by approximately 50% as plasma salicylate concentration increased from 200 to 300 mg/I [17X]. The increase is much more hazardous in toxic levels (181). The concentration dependent binding of disopyramide was first documented in 1974 [182] and has been confirmed by many laboratories [180,183-1851. The free disopyramide fraction at a total concentration of 4.6 pmol,/l is 0.19. but it increases to 0.46 when the total concentration is raised to 18.3 pmol/l [183]. This represents a 2.4-fold increase in the free fraction within the therapeutic range for total disopyramide which is 6-15 pmol/l. Thus, one would expect the disposition of disopyramide to be non-linear when based on total concentration in plasma. and that it would be necessary to consider free disopyramide concentration [186,187]. Protein binding of disopyramide is also altered by competitive binding from its monodealkylated metabolite. The concentrations of the metabolite are highly variable. Among patients receiving enzyme inducing drugs and in patients with renal dysfunction, the metabolite can accumulate to levels higher than its parent compound (25,188) Drug interactions and displacements Drug interaction can be either direct competition by two drugs for the same binding sites, thus resulting in the displacement of the one with weaker binding, or it can be one drug affecting the metabolism and clearance of another drug. Literature on drug interactions is volu~nous but there are only a limited number of clinically significant drug interactions relating to alterations in protein binding. Two of the most clinically significant displacement interactions are the displacement of phenytoin with valproic acid 1189%1941 and warfarin by phenylbutazone or other nonsteroidal anti-inflammatory drugs [195,196]. Valproic acid displaces phenytoin from their common binding site on albumin [194]. It has been shown that this displacement of phenytoin resulted in a 100% increase in the phenytoin free fraction of plasma [194]. As hepatic clearance of phenytoin is dependent on free fraction, the increased free fraction due to displacement will increase phedytoin clearance. The net result should be reduced total phenytoin concentration while the free concentration remains unchanged. Different studies of phenytoin plasma levels following the addition of valproic acid to patients stabilized on phenytoin, however, have shown that total phenytoin levels have either increased or decreased, and that free phenytoin levels have increased [197]. Increased total and free phenytoin levels could be explained if, in addition to displacement. there were inhibition of phenytoin metabolism by valproic acid. This proposed dual mechanism of phenytoin-valproic acid interaction has been complicated by a recent observation that free phenytoin concentration was not altered by valproic acid [19S].

203

The other major ~ticon~lsant drugs, including phenytoin, carbamazepine and phenobarbital, have no effect on valproic acid binding to albumin. The reasons are that valproic acid is more tightly bound to albumin than the other anticonvulsant drugs; that the binding constants for valproic acid, phenytoin and carbamazepine are 6.9 x 104, 1.3 x lo4 and 1.4 x 103, respectively [199]; and also that the molar concentration of valproic acid is higher than those of the other drugs. The displacement of valproic acid by salicylate, in vitro and in vivo, has been demonstrated [200-2023. The mechanism of interaction between these drugs appears to be more than just displacement. The increase in both free valproic acid concentration and half-life of elimination suggest that salicylate inhibits valproic acid metabolism [201]. The highly protein bound anti-coagulant warfarin is dispiaced from albumin binding sites by phenylbutazone f19.51 and other nonsteroidal anti-infl~matory/ analgesic agents including azapropazone, mefenamic acid, naproxen, indomethacin, ketoprofen and iburofen [196]. At therapeutic concentrations, however, only azapropazone and phenylbutazone are known to cause clinically significant potentiation of the anticoagulant effect of warfarin [195,196,203,204]. Relatively few studies have been reported on drug displacement relating to basic drugs which bind not only to albumin but also to &,-acid glycoprotein. The reasons for this may have to do with the fact that albumin binding of cationic drugs is of high capacity and low affinity, and also that the cationic drugs have large volumes of distribution and drug concentrations in plasma water are usually low. Furthermore, binding of the cationic drugs is distributed to more than” one protein (albumin, AAG, lipoproteins), and displacement from one protein may not result in a measurable change in the free fraction of the displaced drug. Thus, even though disopyramide displaces lidocaine and propranolol from AAG, no significant displacement is observed in serum (2051. Other reported drug displacement interactions involving cationic drugs are meperidine and bupivacaine 12061, meperidine and lidocaine [207], and bupivacaine and mepvacaine [208]. Endogenous substances which alter drug binding are free fatty acids and bilirubin, both of which at elevated serum levels are presumed to displace bound drugs from their albumin binding sites. In vitro demonstration of reduced valproic acid binding to serum albumin and patient sera due to increased free fatty acid levels [209-2111 have been confirmed by in vivo studies on patients who are on intravenous fat emulsion therapy and on fasting volunteers [211,212]. A six-fold increase in unbound warfarin and a two-fold increase in unbound phenytoin were produced by increasing plasma free fatty acid concentrations to 2 mEq/l [213]. Other drugs which exhibit reduced binding to albumin in the presence of elevated free fatty acids are the benzodi~epines [120,123,214]. Heparin activates lipoprotein lipase which hydrolyses triglycerides to free fatty acids. It has been reported that the administration of as little as 100 units of heparin (an amount that is likely to enter the bloodstream from an indwelling catheter) produced a 260% increase in unbound diazepam [215]. The high con~ntration of free fatty acid may be artifactual since the degradation of triglycerides continued in vitro after blood collection [216]. This has been confirmed in recent studies which

showed that a large single dose of heparin did not affect the protein binding of prazosin and phenytoin if a lipase inhibitor was present at the time of blood collection [217--2191. In the absence of a lipase inhibitor, the large increases in fatty acids, in vitro, will distort the true extent of drug binding. Bilirubin can also displace drugs from albumin binding sites. High plasma levels of bil~rubin have been reported to have a positive correlation with the free drug fraction of phenytoin and diazepam [124,128,129]. Clinical significance

of altered drug binding

In principle, therapeutic drug monitoring based on the measurement of free drug should be more meaningful than that of total drug because free drug reflects drug concentration at drug receptor sites. Unfortunately, the technology to date for free drug measurement is complex compared to the routine assays for total drug. Hence clinical correlation of drug effects with plasma levels has been based on total drug, and therapeutic ranges are defined for total drug. As long as protein binding of drug is normal and constant (within and between patients~, free drug is a fairly constant fraction of the total drug. As such, measurement of total drug is an adequate estimation of free drug, making routine measurement of free drug unnecessary. Indeed, this has been shown to be the case in a recent report on epileptic patients who were on phenytoin 12201. This relationship between free and total drug does not hold, however, when protein binding is variable or abnormal. In these situations, pharmacological effect may correlate better with free drug and the use of therapeutic ranges based on total drug may be invalid. Altered drug binding due to abnormal protein concentrations, drug interaction and displacement, and concentration-dependent binding is well-documented and summarized in the preceding section, but the clinical significance of altered protein binding has not been studied as thorou~ly. It is clear, however, that nor uil abnormaf drug binding results in free drug concentration which will be of clinital concern. At this juncture it is important to emphasize the distinction between free drug fraction and free drug concentration. Free drugfraczion is the fraction of total drug which is not bound to protein. Free drug ~oncentraf~on is the actual concentration of drug which is not bound to protein. Therefore, depending on the total drug concentration, a normal free drug concentration can actually constitute an abnormal free drug fraction. It should be appreciated that pharmacokinetic principles have predicted that variation in protein binding of drug does not change free drug concentration when steady state is regained at the same rate of drug administration and free drug clearance. Alteration in drug binding, however, does affect total drug clearance. Therefore, total drug concentration at steady state will be different and with it an altered free drug fraction, but the same free drug concentration. Based on this pharmacokinetic consideration, it is evident that there is little clinical significance to many of the reports of altered protein binding resulting from changes in protein concentrations, since free drug clearance in these reports was not affected. Even when free drug concentration was altered as a consequence of altered protein binding, the clinical significance has to be assessed in the context of the width

205

of the therap~tic index of that drug. An altered free phenytoin level will be likely to be a significant change relative to the narrow therapeutic range fur phenytoin. An altered free propranolol level may very well be of little consequence since the therapeutic range for propranolol is wide and not well defined; the minimum concentration for effective beta-bl~kade is ill defined and the patient tolerance for high propranolol levels are well known. Nevertheless, the importance of altered protein binding in the interpretation of serum or plasma drug levels should be emphasized [221]. It is possible to be misled by an abnormal total drug concentration inte~reted in reference to a therapeutic range based on total drug concentration when the free drug concentration has remained therapeutically correct. Such a misinterpretation of total drug levels can lead to unnecessary dosage adjustments to target total drug concentration within the therapeutic range defined for total drug. As a result, free drug concentration may now be inappropriate and the patient may exhibit signs of therapeutic failure or toxicity despite an acceptable total drug concentration. Clinical utility of free drug measurement

Altered protein binding can result in clinically significant alterations in free drug concentration for many patients. In these instances, total drug levels are misleading and free drug levels may be required for optimal patient management. In the case of hypoalbuminemia due to liver disease, reduction in binding to albumin is generally too slow and gradual in onset to be of clinical significance. If free drug clearance is not altered, compensatory mechanisms will increase total drug clearance to maint~n steady state free drug concentration at a lower total drug level. As long as it is recognized that therapeutic range for total drug has been shifted down, neither free drug measurement nor dosage adjustment are necessary. AS liver dysfunc-

tion progresses, the capacity for drug biotransformation is impaired, and free drug clearance eventually will be reduced. Consequently, free drug concentration can no longer be maintained at steady-state level. Thus, for highly protein-bound, restrictively cleared drugs such as phenytoin and valproic acid, advanced liver disease can be accompanied by reduced total drug concentration, increased free drug fraction, and an unpredictable free drug level. In such instances of unpredictability during advanced state of liver disease, measurement of free phenytoin and valproic acid concentrations should be performed. Furthermore, because it is not possible to correlate the extent of liver damage and the onset of compromised free drug clearance, patients with rapidIy progressing liver disease should be monitored by free drug measurement.

Reduction in drug binding in renal disease also lowers steady-state total drug con~ntration and increases free fraction, whereas the free drug ~ncentration will remain unchanged. As a result, therapeutic total drug levels will be lower than the therapeutic range defined for total drug. A number of studies of protein binding of phenytoin and valproic acid in renal patients has shown that free drug fraction is proportional to the degree of renal failure. Therefore, it is possible to calculate at different serum creatinine levels the total drug concentration that will be required to produce therapeutic free drug level. A nomogram can then be constructed to relate

206

total drug therapeutic range to different serum creatinine levels [ 146.149.15 1.2221. While such nomograms are useful as general guidelines. free phenytoin and vatproic acid levels, if available. are the more reliable ways to monitor the complicated uremic patients. An abrupt reduction in protein binding due to drug displacement from binding sites by another drug can lead to a sudden release of bound drug. This is particularly significant if the displacing agent is administered as a bolus by intravenous injection. A transient increase in free drug can be of sufficient magnitude to precipitate an adverse drug reaction, if the drug has a small volume of distribution, a narrow therapeutic index, and is restrictively cleared. The number of~iinjcaf~~ ~~~~n~~cantdrug interactions is smull. The displacement of the anticoagulant warfarin by the nonsteroidal anti-inflammatory agent phenylbutazone and of phenytoin by valproic acid are examples of drug interactions which result in clinically significant toxicity. Addition of valproic acid to patients maintained on phenytoin has precipitated toxicity in at least 25% of the study patients, as well as decreased seizure control in up to 13% [223-2261. In these cases of drug interaction, free drug levels will have better correlation with clinical status than total drug levels. This is particularly evident in the case of phenytoin because of the conflicting observations on free and total phenytoin levels following the addition of valproic acid to the drug regimen, Thus, for patients treated ~vjth Fhen_~toi~l and 15a~pro~~.acid, monitoring of free phenytoin /eve& is recommended, particular@ when an unexpected therupeutic outcome occurs. Concentration-dependent binding of valproic acid and disopyramide should be taken into consideration during optimization of drug therapy for these drugs. As the ratio of free to bound drug varies with total drug concentration, quantitation of total drug concentration is no longer an indirect measure of free drug concentration. This phenomenon is also responsible, at least in part, for the nonlinear plasma total level-dose relationship, although free drug level at steady state is linearly related to dose. Thus, a dosage increase can yield a lower than expected increase in total drug level while free drug level has, in fact, been increased in proportion to dose. Therefore, free drug measurement of drugs such as valproic acid and disopyramide which exhibit variable binding can avoid the misleading information provided by total drug measurement. Moreover, the usefulness of monitoring total disopyramide is even more questionable in view of the protein binding properties of the deakylmetabolite. This metabolite accumulates to levels higher than those of the parent drug in some patients, exhibits concentration dependent binding and competes with disopyramide for binding to protein. Recent studies on the protein binding of cationic drugs suggest that in some acute clinical conditions drug therapy may be more effectively monitored by free drug levels. This may be particularly relevant for cationic drugs which bind to alpha-l acid glycoprotein (AAG) and lipoproteins. The rapidly changing plasma AAG levels after acute tissue injury, such as myocardial infarction and surgery, and the poorly controlled, diet-dependent plasma lipoprotein Ievels, mean that total drug levels should not be the best guide for drug therapy during these critical and unstable periods. Thus free drug monitoring of the cardiac active drugs such as the beta

207

blockers and the calcium channel blockers in the post-infarction period should prove to be important for critical care. Clinical documentation of this is awaiting the results of studies currently under way. Another area where future studies may point to the usefulness of free drug monitoring is perinatal patient care. The physiological changes during labor, delivery and in the immediate post-partum period are accompanied by rapid changes in plasma protein and fatty acid levels. Those medications used during this period which are highly protein bound, such as meperidine, bupivacaine and Valium, will have fluctuating free fractions and free drug concentrations. Obstetricians and anesthesiologists should find free drug monitoring helpful to perinatal care. Laboratory

considerations

Free drug is preferable to total drug monitoring because of its better correlation with pharmacological effects in all patients. Monitoring of total drug, however, is still the standard approach in the clinical laboratories. Free drug monitoring could assume a more important role if there was refinement in the technology to make free drug measurement easier and faster. Routine total drug measurement is technically and operationally simpler than free drug measurement by either equilibrium dialysis or ultrafiltration. Even though equilibrium dialysis has been employed most frequently and has accounted for most of the published information on drug binding, it has the major disadvantage of being laborious and may require many hours or even days to reach equilibrium. Ultrafiltration has been an attractive alternative to equilibrium dialysis. It is even more so now, since previous concerns about the effect of ultrafiltration on binding equilibrium have now been invalidated. Thus, it is not necessary to restrict the ultrafiltration process to give only a small volume of ultrafiltrate, and the analytical problem of measuring low concentration of drug in a small volume of ultrafiltrate is eliminated. Furthermore, centrifugation to obtain ultrafiltrates from multiple specimens is technically simpler and more rapid than equilibrium dialysis, making ultrafiltration more suitable for free drug measurement in the clinical laboratory environment. Conclusion

The indications for free drug monito~ng are understood for a limited number of drugs and clinical situations. Until new and easier techniques for free drug measurement are devised, therapeutic drug monitoring will continue to be based on total drug which will suffice for the management of many but not all patients. The need for free drug monitoring, however, will rise as we advance our understanding of protein binding as well as of the effects of altered binding on drug metabolism and clearance. As clinical experience is gained on the relationship between free drug concentration and drug effects, therapeutic ranges based on free drug rather than total drug concentrations will be established, and informed. clinicians will find free drug m~surement valuable, especially in the management of clinical situations where altered binding can lead to unpredictable free drug concentration, This clinical demand will become the impetus for technological innovations that are

needed activity

to make in clinical

free drug nl~?n~toring laboratories.

the routine

therapeutic

drug

rn~~nlt[~riilg

Acknowledgements I would like to thank Dr. Leslie Shaw for helpful discussions and Ms. Catherine Seneca1 and Ms. Sandra Hengstler for their patience during the preparation of this manuscript. Work in the author’s laboratory was supported by NIH Grant HL-33541. References 1 Koch-Weser 311-316. 2 Koch-Weser 526-531,

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