Free Drug Measurements

One of the fundamental principles of clinical pharmacology is that only “free” drug is pharmacologically active, that is, only the free drug can interact with a receptor to produce a given pharmacologic and therapeutic effect. Measurements of total drug concentration have traditionally been used to estimate clinical response. Clinical experience, as well as many clinical pharmacologic studies, has clearly demonstrated that measurements of drug concentrations in plasma correlate far better with clinical effect than does the drug dose. Because the measurement of total drug concentration (the sum of free drug plus protein-bound drug) is far easier than measurement of free drug concentration, the laboratory generally measures only the total drug concentration. The extent of drug binding to plasma proteins varies widely. The protein-bound fraction varies from less than 10% for caffeine and ethosuximide to greater than 99% for highly bound drugs, such as warfarin and dicumerol.1Changes in the extent of protein binding on the free, pharmacologically active fraction are clinically significant only for highly bound drugs (greater than 80% bound).2,3 For example, if the binding of a drug changes from 98% to 96%, the total drug concentration is unaltered, but the concentration of the free fraction doubles. Knowledge of the free drug concentration is most important in those instances in which the drug is strongly bound by protein, for example, phenytoin, valproic acid, and the tricyclic antidepressants.For many drugs with a high pKa value (phenytoin, primidone, ethosuximide, carbamazepine, etc), the concentration in saliva has been shown to approximate the free drug concentration. This has led to the suggestion that in many instances saliva should be substituted for the plasma sample. Since saliva is collected by noninvasive techniques, there are further advantages to this approach.4,5While the routine measurement of free drug concentration may be desirable, it has not been achieved in practice. Equilibrium dialysis is time-consuming and requires fairly large sample volumes, and the numerous membranes available commercially that allow free drug to be measured in the ultrafiltrate are costly. Free drug concentrations can be measured by using either equilibrium dialysis or, more commonly, devices with molecular cutoff filters, such as the Amicon Centrifree micropartition system or the Worthington Diagnostics “ultrafree” system.Temperature affects the degree of binding of phenytoin to albumin, with drug binding decreasing as temperature increases. Control of temperature is therefore important. A 10-degree drop in temperature (37°C to 27°C) can result ina 25% decrease in the “free fraction.” 6 As the patient's temperature is usually 37°C, it is recommended to use this temperature when assessing free phenytoin concentrations. The free concentrations of phenytoin have been shown to be increased significantly in patients with human immunodeficiency virus.7 It should be noted that the temperature effect on percent free drug varies from drug to drug. For example, it is minimal for carbamazepine.Albumin is quantitatively the most important binding protein for many drugs, including warfarin, phenytoin, and valproic acid.2 In addition to albumin, various blood constituents, such as red blood cells and α1-acid glycoprotein are capable of binding drugs. The concentration in plasma of α1-acid glycoprotein, a protein which binds many basic drugs, increases with infections, inflammations, and in malignant diseases. For this reason, it may be important to measure free drug concentration in patients receiving propranolol, chlorpromazine, lidocaine, or disopyramide, all of which bind to α1-acid glycoprotein.8,9In my experience, clinician requests for free drug measurement occur most frequently with phenytoin. As already mentioned, phenytoin is strongly (90%) protein bound. Any disease causing a decrease in serum protein binding of phenytoin can be associated with a large increase in free phenytoin concentration, leading to phenytoin toxicity. Also, numerous agents (valproic acid, salicylates, thiazides, and endogenous compounds in patients with renal failure) displace phenytoin from its binding site on albumin. In the presence of these compounds, the free fraction of phenytoin increases, placing the patient at increased risk for toxicity, and symptoms of toxicity become apparent. These patients have signs of phenytoin toxicity at serum concentrations in the therapeutic range (10–20 mg/L). In these instances, the free phenytoin concentration should be adjusted to provide a free phenytoin concentration between 1 and 2 mg/L.4Carbamazepine is metabolized by carbamazepine epoxidase to carbamazepine-10,11-epoxide, a metabolite that has significant anticonvulsant activity and that is approximately equipotent to the parent drug. Carbamazepine is bound to plasma proteins (65% to 80% bound), while the epoxide binding is less strong (50% to 60% bound). Because the free fraction is pharmacologically active and because the epoxide has a greater percentage in the free form and is equipotent to the parent drug, epoxide presumably contributes significantly to the anticonvulsant effect. Two recent articles emphasize the importance of measuring carbamazepine epoxide in patients receiving either valproic acid or lamotrigine.10,11The protein binding of valproic acid is variable and depends on many factors, including the concentration of valproic acid in serum (eg, at 10–60 mg/L there is approximately 5% free drug, while at 145 mg/L there is approximately 20% free drug). Measurement of the free valproic acid concentration may therefore be requested occasionally.4Although digoxin is only slightly protein bound (20% to 40%), measurement of free digoxin is being requested increasingly in patients receiving either Fab antibodies, such as Digibind, for treatment of digoxin toxicity or in patients with digoxin-like immunoreactive factors (DLIFs), such as neonates, patients with renal or liver disease, and women in the third trimester of pregnancy being treated with digoxin.4 Digoxin-like factors are strongly bound by protein, and measurement of digoxin in the ultrafiltrate provides the free digoxin concentration and separates it from the cross-reactive DLIFs. In this way, the physician can evaluate the patient's digoxin status even in the presence of Digibind or DLIFs. By multiplying the conventional therapeutic range of 0.8to 2.0 ng/mL by 0.8, one arrives at a therapeutic range for free digoxin of 0.6 to 1.6 ng/mL. Finally, patients receiving Digibind will have very high plasma or serum concentrations of digoxin when measured by most methods. This is because the Fab antibody Digibind draws digoxin out of skeletal muscle and heart tissue. This Fab-bound digoxin is not pharmacologically active digoxin, and for this reason measurement of free digoxin in the ultrafiltrate is strongly recommended.12–16