Altered Plasma Protein Binding Research Articles

Facing the Facts of Altered Plasma Protein Binding: Do Current Models Correctly Predict Changes in Fraction Unbound in Special Populations?

Accounting for variability in plasma protein binding of drugs is an essential input to physiologically-based pharmacokinetic (PBPK) models of special populations. Prediction of fraction unbound in plasma (fu) in such populations typically considers changes in plasma protein concentration while assuming that the binding affinity remains unchanged. A good correlation between predicted vs observed fu data reported for various drugs in a given special population is often used as a justification for such predictive methods. However, none of these analyses evaluated the prediction of the fold-change in fu in special populations relative to the reference population. This would be a more appropriate assessment of the predictivity, analogous to drug-drug interactions. In this study, predictive performance of the single protein binding model was assessed by predicting fu for alpha-1-acid glycoprotein and albumin bound drugs in hepatic impairment, renal impairment, paediatric, elderly, patients with inflammatory disease, and in different ethnic groups for a dataset of >200 drugs. For albumin models, the concordance correlation coefficients for predicted fu were >0.90 for 16 out of 17 populations with sub-groups, indicating strong agreement between predicted and observed values. In contrast, concordance correlation coefficients for predicted fold-change in fu for the same dataset were <0.38 for all populations and sub-groups. Trends were similar for alpha-1-acid glycoprotein models. Accordingly, the predictions of fu solely based on changes in protein concentrations in plasma cannot explain the observed values in some special populations. We recommend further consideration of the impact of changes in special populations to endogenous substances that competitively bind to plasma proteins, and changes in albumin structure due to posttranslational modifications. PBPK models of special populations for highly bound drugs should preferably use measured fu data to ensure reliable prediction of drug exposure or compare predicted unbound drug exposure between populations knowing that these will not be sensitive to changes in fu.

Benefits, safety and side effects of tumescent local anesthesia in dermatologic surgery in infants.

Tumescent local anesthesia (TLA) plays an important role in dermatosurgical procedures. TLA has several benefits such as long-lasting anesthesia, reduced bleeding during surgery and the avoidance of general anesthesia-associated complications. Convenience and a favorable risk profile along with a broad spectrum of indications are further reasons why TLA is increasingly applied in infants as well. There are not only a variety of indications for surgical excisions in infancy, such as congenital nevi, but also substantial benefits when performing these excisions at an early age. These include the smaller size of the lesions as well as the unproblematic wound healing and tissue regeneration in infancy. Nevertheless, several aspects need to be considered when applying TLA in infants including dosing, altered plasma protein binding and the need for adequate and long-lasting pain control.

Short-Term Fasting Alters Pharmacokinetics of Cytochrome P450 Probe Drugs: Does Protein Binding Play a Role?

Background and ObjectivesShort-term fasting differentially alters cytochrome P450 (CYP) mediated drug metabolism. This has been established by using CYP-enzyme selective probe drugs. However, the observed effects of fasting on the pharmacokinetics of these probe drugs may also include the effects of altered plasma protein binding of these drugs. Therefore, we studied the effect of short-term fasting on protein binding of five commonly used probe drugs [caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A4), omeprazole (CYP2C19) and S-warfarin (CYP2C9)].MethodsThe free and total plasma concentrations of the five probe drugs were analyzed by LC–MS/MS in samples retrieved in a cross-over study in which nine healthy subjects received an intravenous administration of the cocktail after an overnight fast (control) and after 36 h of fasting.ResultsShort-term fasting increased plasma free fatty acid concentrations from 0.48 mmol/L (control) to 1.29 mmol/L (36 h fasting) (p = 0.012). Short-term fasting did not alter the free fractions of caffeine, metoprolol and omeprazole compared to the control intervention (p > 0.05). Power to detect a difference for midazolam and S-warfarin was low since the majority of free concentrations were below the limit of quantification.ConclusionsThis study demonstrates that short-term fasting does not alter protein binding of the probe drugs caffeine, metoprolol and omeprazole.Electronic supplementary materialThe online version of this article (doi:10.1007/s13318-017-0437-7) contains supplementary material, which is available to authorized users.

A Physiologically Based Pharmacokinetic Perspective on the Clinical Utility of Albumin-Based Dose Adjustments in Critically Ill Patients.

In hypo-albuminemia, the extent of albumin binding of a drug decreases. The resulting change in plasma protein binding only rarely leads to clinically relevant changes in unbound drug exposure. Nevertheless, in the critically ill, a tendency to increase dosing of anti-infective therapy is seen in patients experiencing hypo-albuminemia. To reconcile basic pharmacological principles with current clinical practice, this work presents a pharmacologically-based pharmacokinetic simulation study to emphasize the (lack of) effect of altered plasma protein binding on a drug's concentration-time profile and associated pharmacokinetic parameters. Four virtual compounds, representing a broad chemical space (low/high clearance/volume of distribution), were created and administered to a virtual population of normal patients and three types of hypo-albuminemic patients in Simcyp®. The influence of decreased plasma protein binding in hypoalbuminemia on the pharmacokinetic parameters and profiles of these four compounds was investigated. Simulation results showed that while high-clearance compounds suffer from increased unbound exposure with decreased plasma protein binding, the unbound exposure of low-clearance compounds was unaffected. However, for the subset of low-clearance compounds with a small volume of distribution, it appeared that there were still alterations in their plasma concentration-time profiles. Since this may lead to different times above a minimum inhibitory concentration value, this might affect the bacterial killing for some anti-infective drugs. Overall, for any compound involved in the simulations, the unbound exposure did not decrease in plasma protein binding subjects relative to normal plasma protein binding subjects. This finding is in line with the few case-controlled studies in the literature. Hence, increasing the dose/dosing frequency seems futile and might reduce the benefit-risk ratio for narrow therapeutic index drugs. Moreover, these simulations indicate that when only total plasma concentrations and derived pharmacokinetic parameters are considered, incorrect conclusions will be drawn.

Drug–Drug Interactions Related to Altered Absorption and Plasma Protein Binding: Theoretical and Regulatory Considerations, and an Industry Perspective

Drug-drug interactions (DDIs) related to altered drug absorption and plasma protein binding have received much less attention from regulatory agencies relative to DDIs mediated via drug metabolizing enzymes and transporters. In this review, a number of theoretical bases and regulatory framework are presented for these DDI aspects. Also presented is an industry perspective on how to approach these issues in support of drug development. Overall, with the exception of highly permeable and highly soluble (BCS 1) drugs, DDIs related to drug-induced changes in gastrointestinal (GI) physiology can be substantial, thus warranting more attentions. For a better understanding of absorption-associated DDI potential in a clinical setting, mechanistic studies should be conducted based on holistic integration of the pharmaceutical profiles (e.g., pH-dependent solubility) and pharmacological properties (e.g., GI physiology and therapeutic margin) of drug candidates. Although majority of DDI events related to altered plasma protein binding are not expected to be of clinical significance, exceptions exist for a subset of compounds with certain pharmacokinetic and pharmacological properties. Knowledge of the identity of binding proteins and the binding extent in various clinical setting (including disease states) can be valuable in aiding clinical DDI data interpretations, and ensuring safe and effective use of new drugs.

Physiologically‐Based Pharmacokinetic (PBPK) Modeling and Simulations Principles, Methods, and Applications in the Pharmaceutical Industry

CPT: Pharmacometrics &amp; Systems Pharmacology (2013) 2, e55; doi:10.1038/psp.2013.29; advance online publication 10 July 2013

Calculation of Normalized Drug Concentrations in the Presence of Altered Plasma Protein Binding

In many clinical situations, measurement of the total drug concentration does not provide the needed information concerning the fraction of unbound drug in plasma, which is available for pharmacodynamic action. To address this, a 'normalized concentration' can be calculated on the basis of the observed total drug concentration and the serum protein level. Up to now, this method has only been applied to phenytoin. Several equations for calculating normalized concentrations of phenytoin have been published, many leading to different results. Regrettably, all of the equations in the current literature are based on an outdated model of drug binding to human serum albumin and are based on the fraction of unbound drug, which is known to depend on both protein and drug concentrations. In response to the relatively new scientific evidence about drug binding to human plasma proteins, the objective of the present study is to develop a general method for calculating normalized drug concentrations in the presence of altered plasma protein binding. When several drug molecules can be bound by a protein molecule, multiple equilibria are established; these equilibria may be formulated in terms of a stoichiometric analysis or a site-oriented analysis. Both models are currently encountered in the scientific literature, sometimes without clear identification of which model is used. The present study presents the basic equations for both models and shows how the normalized concentration can be calculated on the basis of the measured drug concentration, the protein level and the binding constants. The normalized concentration can be calculated for any drug, using the same simple equation regardless of the binding model and the number of binding proteins. Explicit solutions are presented for particular cases of clinical importance. The new model is validated by comparison with the Winter-Tozer equation for calculating the normalized phenytoin concentration and is found to be equivalent for concentrations close to therapeutic concentrations. In the case of phenytoin, the main advantage of the new equation is that it also works outside the linear binding range. A new comprehensive method for calculating normalized drug concentrations is developed, allowing drug concentrations to be interpreted correctly in cases of altered drug-protein binding. The calculations are based on binding constants and are applicable to any protein level and drug concentration, without being limited to linear binding of drugs to proteins. The new model is expected to become important in pharmacokinetic-pharmacodynamic modelling, allometric scaling and population pharmacokinetics because it provides the ability to accurately take into account physiological and pathological changes in protein binding. As a direct clinical application, the equations can be used to calculate normalized drug concentrations in patients with abnormal protein levels, such as the elderly, trauma patients and paediatric patients.

The binding of the antimalarial arteether to human plasma proteins in-vitro.

The binding of the novel antimalarial drug, arteether, to human plasma, pure albumin and alpha 1-acid glycoprotein has been investigated by ultrafiltration, using [14C]arteether. The protein binding in plasma obtained from 11 healthy male subjects ranged from 73.4 to 81.8% bound, with a mean of 78.7 +/- 2.1%. The binding of drug in plasma was mainly accounted for by binding to albumin and alpha 1-acid glycoprotein. Scatchard analysis of the binding data revealed that the binding affinity of arteether to alpha 1-acid glycoprotein is much greater (20-fold) than that to albumin. This suggests that alpha 1-acid glycoprotein is the more important binding protein in plasma. This may have clinical importance due to alterations in plasma protein binding in patients with malaria, as the concentration of alpha 1-acid glycoprotein is markedly increased during malarial infection.

Effect of altered AGP plasma binding on heart rate changes by S(−)‐propranolol in rats using mechanism‐based estimations of in vivo receptor affinity (KB,vivo)

In contrast to the impact of plasma protein binding on pharmacokinetics, no quantitative in vivo information is available on its impact on pharmacodynamics. The pharmacokinetic–pharmacodynamic relationship of the model drug S(−)‐propranolol was evaluated using mechanism‐based estimations of in vivo receptor affinity (KB,vivo), under conditions of altered plasma protein binding resulting from different levels of alpha‐1‐acid glycoprotein (AGP). Male Wistar Kyoto rats with isoprenaline‐induced tachycardia received an intravenous infusion of S(−)‐propranolol, on postsurgery day 2 (n = 7) and day 7 (n = 8) with elevated and normal plasma protein binding, respectively. Serial blood samples were taken in parallel to heart rate measurements. AGP concentrations at 2 and 7 days postsurgery were 708 ± 274 and 176 ± 111 µg/mL (mean ± SE), respectively. Using nonlinear mixed effects modeling, AGP concentration was a covariate for intercompartmental clearance for the third compartment of the pharmacokinetic model of S(−)‐propranolol. Individual values of AGP concentrations ranged between 110 and 1150 µg/mL, and were associated with KB,vivo values of S(−)‐propranolol from 7.0 to 30 nM. Using the KB,vivo for S(−)‐propranolol with correction for average values for normal and elevated plasma protein binding, nearly identical values were found. This confirms, strictly quantitative, earlier indications that plasma protein binding restricts the pharmacodynamics of S(−)‐propranolol. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2511–2520, 2010

Effect of the cardiopulmonary bypass procedure during cardiac surgery on CYP2C9 activity in human

Clinical observations suggest that warfarin sensitivity is increased in patients undergoing cardiac surgery that involves a cardiopulmonary bypass (CPB) procedure. Factors such as hemodilution, altered plasma protein binding or change in visceral perfusion during CPB may cause changes in pharmacokinetic parameters of various drugs. Since CYP2C9 is the major enzyme that metabolizes warfarin, we aimed to investigate if the increase in warfarin sensitivity was associated with inhibition of the activity of this individual enzyme due to CPB in human. Losartan oxidation to E3174 was used as a marker for CYP2C9 activity. Urinary losartan/E3174 ratios after a single 25 mg oral dose were measured in 23 patients at three occasions: at the baseline, and on the first and fifth postoperative days. The average metabolic ratio (median, range) was significantly increased for about 3.3 fold as compared to the baseline value (0.84, 0.1–7.94 vs. 2.80, 0.7–278.2) in the acute phase immediately after the surgery (P=0.01). The metabolic ratio returned to the baseline value when measured at the fifth postoperative day (P=0.24). These results suggest that CYP2C9 metabolic activity is inhibited at the very early stage of postoperative period and recovers as early as within five days in patients that undergo cardiac surgery with a cardiopulmonary bypass procedure. (Supported by TUBITAK-SBAG COST B25-105S027 and UY/TUBA-GEBIP/2005-17)

Changes in the effect of propofol in response to altered plasma protein binding during normothermic cardiopulmonary bypass

During normothermic cardiopulmonary bypass (CPB), the effect on propofol pharmacokinetics of changes in its binding to plasma proteins is consistent with the predictions of the well-stirred model of hepatic elimination for nonrestrictively cleared drug. However, whether changes in binding lead to clinically significant changes in the drug effect remains unclear. The purpose of this study was to assess changes in the drug effect of propofol in response to altered plasma binding using quantitative EEG measurements. Thirty patients undergoing cardiac surgery were assigned randomly to receive propofol infusions at 4 (Group P-4) or 6 (Group P-6) mg kg(-1) h(-1) during surgery. The concentration of propofol in blood samples, collected from the radial artery at predetermined intervals, was determined by HPLC. The unbound fraction of drug in plasma was estimated using equilibrium dialysis. Bispectral index (BIS) and burst suppression ratio (BSR) were measured at the time blood samples were collected. The total concentration of propofol in blood was unchanged during CPB relative to the pre-CPB value in both groups. However, the fraction of unbound propofol in blood increased by 2-fold during CPB. While BIS values were unchanged during CPB in Group P-4, there was a slight, but significant, decrease in Group P-6. In both groups, BSR significantly increased during CPB. BIS values showed a weak correlation with the concentration of unbound propofol (r(2)=0.19, P<0.001). BSR showed a moderate correlation with the concentration of unbound propofol (r(2)=0.56, P<0.001). The anaesthetic effect of propofol significantly increased during CPB without any alteration in the total drug concentration. The enhanced efficacy may be caused by a reduction in plasma binding of the drug.

Drug development and use in the elderly: search for the right dose and dosing regimen

Across the globe over the last few decades, there has been a remarkable increase in the proportion of the population which is elderly, and this trend is set to continue. Data from 10 countries that accounted for about 55% of the world population of 6079 million in the year 2000 provide an overview of this significant shift in demography. Table 1 summarizes the projections for the next 25 years. Hitherto, increases in life expectancy have been most obvious in the affluent countries; however, improvements in public health and disease control are also leading to increases in life expectancy in the less affluent countries. As a result of relatively larger increases in life expectancy at birth, significant shifts in demography are also expected in countries that are at present economically less affluent. The number of elderly people in the world is therefore expected to continue to increase for some considerable time. Interestingly, the most dramatic increases in the proportion of the population who are elderly are anticipated in countries where total populations are projected to decrease (such as Japan, Germany and Italy). Table 1 Age-related demography in 2000 and projected changes in 10 major countries The 2001 population census of the UK revealed that since the census of 1951, the proportion of elderly population above 65 years and 85 years has increased from 16% and 0.4% respectively to 21% and 1.9%, respectively. These changes in demography will have significant effects on the economics generally as well as on the provisions for healthcare. Cardiovascular and neurological diseases and cancers are the most prevalent in the elderly. These three groups of diseases account for about 54% of the total burden of disease in Europe in terms of disability adjusted life years. Of the total gross Hospital and Community Health Services expenditure of £31.9 billion in the year 2001–2002 in the UK, 13% was expended on people aged 65–74 years, 16% on those aged 75–84 years and 10% for people aged 85 years or more. Furthermore, about 55% of the community prescriptions during 2001 in the UK were dispensed for the elderly. Cardiovascular and psychoactive drugs accounted for more than 40% of these prescriptions. In view of many age-related changes in the pharmacokinetics and pharmacodynamics of a drug, the safe and effective prescribing of medicines in the elderly will continue to present a major challenge. This review provides an overview of the issues relevant to development and clinical use of drugs in the elderly population, with particular reference to determining the right dosage and dose regimen and the regulatory requirements that facilitate this process. It also examines whether, as a result of advancing age per se, the dosing regimens in the elderly and the frail elderly might be different from those in the non-elderly.

Therapeutic drug monitoring as a tool in treating HIV infection.

Despite its success antiretroviral therapy (ART) is still associated with a significant risk of toxicity and subsequent virological failure. Pending the introduction of drugs with greater potency and durability one of the most pressing priorities in HIV treatment is to identify ways in which to maximize the efficacy of existing therapeutic options while minimizing its toxicity. Resistance testing has been shown to be one such strategy. Therapeutic drug monitoring (TDM) for HIV drugs has been widely proposed as another tool by which treatment may be optimized. There are some parallels with resistance testing not least the potential difficulties or pitfalls in interpreting test results and the understandable pressure for provision of this service in the face of increasingly limited treatment options notwithstanding the current lack of data from controlled clinical studies. In this reviews we shall discuss the value of TDM in the context of a ‘monitoring service (in other words drug concentrations are provided in conjunction with interpretation and advice) rather than a facility for ‘measuring drug concentrations. This distinction is important since the interpretation of drug concentrations is fraught with complexity. (excerpt)

Age-related changes in protein binding of drugs: implications for therapy.

The plasma protein binding of drugs, particularly those that are highly bound, may have significant clinical implications. Although protein binding is a major determinant of drug action, it is only one of a myriad of factors that influence drug disposition. The extent of protein binding is a function of drug and protein concentrations, the affinity constant for the drug-protein interaction and the number of protein binding sites per class of binding site. Age-related changes in protein binding are usually not clinically important in drug therapy. Albumin levels are generally decreased in the elderly, whereas alpha1-acid glycoprotein levels are not altered by age per se. Alterations in plasma protein binding that occur in the elderly are generally not attributed to age, but rather to physiological and pathophysiological changes or disease states that may occur more frequently in the elderly and most often account for altered protein binding. Age-related physiological changes, such as decreased renal function, decreased hepatic function and decreased cardiac output, generally produce more clinically significant alterations in drug disposition than that seen with alterations in drug plasma protein binding. An understanding of the inter-relationships between drug concentrations, protein binding, the physiology of aging, disease, pharmacokinetics and pharmacodynamics is necessary for effective therapeutic monitoring. Monitoring of unbound drug concentrations simplifies these relationships and provides the fundamental information needed for dosage regimen development and adjustment. Drug therapy in the elderly should be individualised taking into account all of these factors.

The pharmacokinetics of anesthetic drugs and adjuvants during cardiopulmonary bypass.

The institution of cardiopulmonary bypass during cardiac surgery has profound effects on the plasma concentration of drugs and thus their therapeutic effectiveness. These changes occur through acute hemodilution, altered plasma protein binding, hypotension, as well as the use of hypothermia and heparin administration. Isolation of the lungs from the circulation and the possible sequestration of drugs in the bypass circuit also affect drug plasma concentrations on bypass. The individual characteristics of the drug in question are also important in determining the final plasma concentration: Lipid soluble drugs with a high volume of distribution may be more readily taken up by bypass equipment, but the initial fall in concentration at the start of cardiopulmonary bypass may be more readily counteracted by back diffusion into plasma, if large tissue stores have accumulated. The extent of the drug's plasma protein binding is of importance as the effective free fraction in plasma for highly bound drugs will be sensitive to changes in plasma protein binding brought on by factors such as hemodilution, heparin administration as well as alpha, acid-glycoprotein binding. Clearly the fate of drugs administered before or on bypass is complex and can only be accurately determined by specific studies evaluating drug plasma concentrations. This review updates the available data on anesthetics and drugs used during cardiac surgery in order that anesthetists may predict better the likely effect of drugs administered before or during cardiopulmonary bypass.

Tenoxicam Pharmacokinetics In Rats: A Population Model

This study was designed to establish the in vivo relationship between tenoxicam disposition and changes in plasma protein binding measured as an unbound fraction in plasma (fu). Tenoxicam was administered as a bolus 5-mg/kg dose, and total plasma concentrations, plasma albumin percentage, and fu were examined in five groups of rats (uremia or anephric states were experimentally induced in four groups to decrease plasma protein levels). Albumin levels were significantly decreased in all experimentally altered groups with respect to control animals (p < 0.01). A two-compartment population pharmacokinetic model that includes the effect of fu on the kinetic parameters was proposed to describe tenoxicam plasma concentration profiles. Plasma clearance (CL) increased but not proportionally with fu. Apparent volume of distribution of the central compartment (v) was linearly related to changes in fu, and intercompartmental clearance was not affected by altered plasma protein binding. Expressing pharmacokinetic parameters as a function of fu resulted in a three- and five-fold decrease in the variability associated with CL and V, respectively.

Pharmacokinetics of nordiazepam in physical dependence and precipitated abstinence in dogs

Previous studies suggested that the extensive accumulation of benzodiazepines is an important factor in the induction of physical dependence. The mechanistic basis for accumulation of nordiazepam (ND) and its metabolite, oxazepam (OX), have been examined in crossover studies in drug-naive and in ND-dependent dogs that exhibited a flumazenil-precipitated abstinence syndrome. ND and parent OX have similar pharmacokinetic profiles. Steady-state plasma levels of ND and OX cannot be predicted from single-dose pharmacokinetics. Reduced plasma clearance of ND and altered plasma protein binding were observed in dogs physically dependent upon ND. The benzodiazepine antagonist, flumazenil. significantlyreduces steady-state plasma levels of total and free ND.

Risks of pregnancy in women with epilepsy.

Pregnant women with epilepsy are at increased risk of seizures and complications. An increase in seizure frequency is seen in 25-30% of pregnant women with epilepsy; the offspring of mothers who experienced seizures during pregnancy are at a 2.5 times higher risk for seizures later in life. One of the main reasons for the increase in seizures during pregnancy is a decline in plasma concentrations of antiepileptic drug (AED) that occurs as pregnancy progresses, largely as a result of marked alterations in plasma protein binding. It is well known that epilepsy represents a risk for a variety of adverse pregnancy outcomes or malformations, especially in polytherapy. The adverse outcomes range from dysmorphic features to hemorrhagic disorders resulting from a deficiency of vitamin K-dependent clotting factors or to spina bifida. Folic acid supplements appear to reduce the risk of spina bifida. A strong genetic link seems to exist for many of the malformations that occur, and more research is required in this field. In the meantime, there are interventions that clinicians can already make to reduce the risk of adverse outcomes, such as seizure control without toxicity, monotherapy, and preconceptual use of vitamins with folate.

Severe Phenytoin Intoxication as a Result of Altered Protein Binding in Aids

Alterations in plasma protein binding may alter patient response to pharmaceutical agents because only free drug is considered to be pharmacologically active. Such alterations appear to be more significant with highly bound agents such as phenytoin. Traditionally, most drug assays monitor total drug concentrations and do not quantitate free drug. When binding alterations are present, total drug concentrations may mislead clinicians in evaluating patient response. We describe a case in which profound hypoalbuminemia (0.2 g/dL), associated with focal segmental glomerulosclerosis, produced toxic free phenytoin concentrations (4.9 micrograms/mL) in an HIV-positive 25-year-old black woman. At such a high serum concentration of free phenytoin, the patient exhibited seizure-like effects. Renal abnormalities and hypoalbuminemia associated with acquired immunodeficiency syndrome (AIDS) may place patients at risk for elevated free fractions of phenytoin and subsequent toxicity.

Pharmacokinetics of anticonvulsants in pregnancy: Alterations in plasma protein binding

Anticonvulsant levels decline as pregnancy progress, even in the face of constant and, in some cases, increased dosages of medications. It has been suggested that this decline is responsible for the increase in seizure frequency seen in approximately one-third of the women with epilepsy who become pregnant. Changes in plasma protein binding may explain the declines in anticonvulsant concentrations during pregnancy. A prospective cohort study was designed to test this hypothesis. Carbamazepine, phenytoin and phenobarbital were studied. The mean total concentrations of all 3 drugs declined as pregnancy progressed, rising in the postpartum period. Free concentrations also declined, but did so significantly only for phenobarbital. The free fraction for all anticonvulsants studied rose significantly throughout pregnancy. Protein binding is significantly altered during pregnancy for all 3 drugs studied and appears to account for much of the decline in anticonvulsant concentrations seen in this condition. It is suggested that free rather than total drug concentrations be monitored in pregnant women with epilepsy.