Free Drug Monitoring Research Articles

Analysis of free concentrations of lamotrigine and active oxcarbazepine metabolite in clinical patients by hollow-fiber centrifugal ultrafiltration.

Aim: To establish a simple and accurate method to explore the correlation between free and total concentrations of lamotrigine (LTG) and the active oxcarbazepinemetabolite monohydroxy derivative (MHD) (10,11-dihydro-10-hydroxycarbamazepine) in clinical patients. Materials & methods: Serum samples were prepared by hollow-fiber centrifugal ultrafiltration and then injected into UPLC for analysis. Results: Absolute recovery was as high as approximately 90.1-98.6% with excellent precision (relative standard deviation <6.7%). Analysis time was reduced to 5min. There were significant individual differences in the protein binding rates of both LTG and MHD that were probably due to the use of different clinical patients. Conclusion: Free concentrations of LTG and MHD cannot be estimated by total concentration in specific clinical patients. Free drug monitoring of LTG and MHD in clinical therapeutic drug monitoring is important and essential.

Validated LC-MS/MS method for quantitation of total and free mycophenolic acid concentration and its application to a pharmacokinetic study in pediatric renal transplant recipients.

A simple and sensitive LC-MS/MS method was established to quantify total and free mycophenolic acid (MPA) plasma concentrations during immunosuppressive medication for pediatric renal transplantation. The chromatographic separation was performed with the Hypersil GOLD C18 column, using a mobile phase consisting of 0.1% formic acid in water and acetonitrile (60:40, v/v) at an isocratic flow rate of 0.4 ml/min. An Agilent 6420 triple quadrupole mass spectrometer was operated via a positive electrospray ionization interface using the transitions m/z 321.14 → 206.9 for MPA and m/z 324.15 → 209.9 for MPA-d3 (internal standard). The linearity was 0.1-50 μg/ml for total MPA and 0.0025-0.5 μg/ml for free MPA. The within-run and between-run precisions were all <5% and accuracy was within 96.23-107.63%. The validated method was successfully aspplied to a pharmacokinetic study in 28 pediatric renal recipients. The mean free fraction of MPA in our patients was 0.89% (ranging from 0.62 to 1.25%) and albumin level played a major role in the variability of free fraction of MPA, thus, in pediatric patients with hypoproteinemia, close free drug monitoring and dose adjustments should be considered to prevent toxicity.

Simultaneous Determination of Protein-Unbound Cyclosporine A and Mycophenolic Acid in Kidney Transplant Patients Using Liquid Chromatography–Tandem Mass Spectrometry

Therapeutic drug monitoring (TDM) of immunosuppressants is essential to optimize patient care after organ transplantation. In blood, most immunosuppressive drugs are bound to plasma proteins or located inside blood cells. However, it is generally assumed that only protein-unbound (free) drug concentrations are pharmacologically active and could therefore better reflect the clinical outcome. Study data are still limited due to lacking rapid analytical methods. Therefore, a simple multiplex method for direct measurement of free cyclosporine A (CsA) and mycophenolic acid (MPA) has been developed. The sample preparation included ultracentrifugation, followed by liquid-liquid extraction. Stable isotope labeled analogues of CsA and MPA were used as internal standards. The LC-MS/MS analysis was performed on a triple quadrupole mass spectrometer in the multiple reaction monitoring mode. The validated assay was used in a study of 40 blood samples from kidney transplant patients. The lower limits of quantification were 0.1 (CsA) and 0.5 ng/mL (MPA). Assay linearity was confirmed in the concentration ranges of 0.1-10.0 ng/mL (CsA) and 0.5-100 ng/mL (MPA). For both analytes, inaccuracy was ≤9.8% and imprecision was ≤7.8%. The extraction efficiency ranged between 91% and 96%. In the patient samples the average free CsA and MPA fractions were 5.8% (2.1%-16.8%) and 1.2% (0.5%-2.4%) respectively. A reliable and highly sensitive LC-MS/MS method as a new suitable tool for measuring protein-unbound CsA and MPA has been developed, validated and applied in kidney transplant patient samples. Now, larger studies can be conducted to investigate the benefit of free drug monitoring in transplant recipients.

PETINIA Total Phenytoin Assay on the Vista 1500 Analyzer Is Not Suitable for Monitoring Free Phenytoin Concentration if Ultrafiltration Is Performed at Room Temperature

Phenytoin is a strongly protein-bound anticonvulsant requiring free drug monitoring for certain patients. Recently, free phenytoin assay on the TDx analyzer was discontinued by the Abbott Laboratories, but Roche Diagnostics has an assay for the determination of free phenytoin. Because a clinical laboratory may not have an instrument for using Roche free phenytoin assay, we studied the possibility of determining free phenytoin using particle-enhanced turbidimetric inhibition immunoassay (PETINIA) total phenytoin assay on the Vista 1500 analyzer by comparing free phenytoin values obtained by Roche assay and Hitachi 917 analyzer with values obtained by using PETINIA assay. When protein-free ultrafiltrates were prepared at room temperature from 41 different patients taking phenytoin, using Roche assay (x axis) and PETINIA (y axis) assay, the following regression equation was observed: y = 0.8526x + 0.0661 (r = 0.85, n = 41). The regression equation indicated significant negative bias with the reference method. However, when additional 30 specimens were studied where ultrafiltration was performed at 37°C, lesser negative bias was observed as revealed by the regression equation: y = 0.922x - 0.117 (r = 0.90, n = 30). We conclude that PETINIA assay on the Vista 1500 analyzer is not suitable for monitoring free phenytoin concentration if ultrafiltration is performed at room temperature but may be used with caution if ultrafiltration is performed at physiological temperature of 37°C, which is the desirable temperature for preparation of ultrafiltrate.

Therapeutic Drug Monitoring of the Newer Anti-Epilepsy Medications.

In the past twenty years, 14 new antiepileptic drugs have been approved for use in the United States and/or Europe. These drugs are eslicarbazepine acetate, felbamate, gabapentin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, pregabalin, rufinamide, stiripentol, tiagabine, topiramate, vigabatrin and zonisamide. In general, the clinical utility of therapeutic drug monitoring has not been established in clinical trials for these new anticonvulsants, and clear guidelines for drug monitoring have yet to be defined. The antiepileptic drugs with the strongest justifications for drug monitoring are lamotrigine, oxcarbazepine, stiripentol, and zonisamide. Stiripentol and tiagabine are strongly protein bound and are candidates for free drug monitoring. Therapeutic drug monitoring has lower utility for gabapentin, pregabalin, and vigabatrin. Measurement of salivary drug concentrations has potential utility for therapeutic drug monitoring of lamotrigine, levetiracetam, and topiramate. Therapeutic drug monitoring of the new antiepileptic drugs will be discussed in managing patients with epilepsy.

Therapeutic Drug Monitoring of the Newer Anti-Epilepsy Medications

In the past twenty years, 14 new antiepileptic drugs have been approved for use in the United States and/or Europe. These drugs are eslicarbazepine acetate, felbamate, gabapentin, lacosamide, lamotrigine, levetiracetam, oxcarbazepine, pregabalin, rufinamide, stiripentol, tiagabine, topiramate, vigabatrin and zonisamide. In general, the clinical utility of therapeutic drug monitoring has not been established in clinical trials for these new anticonvulsants, and clear guidelines for drug monitoring have yet to be defined. The antiepileptic drugs with the strongest justifications for drug monitoring are lamotrigine, oxcarbazepine, stiripentol, and zonisamide. Stiripentol and tiagabine are strongly protein bound and are candidates for free drug monitoring. Therapeutic drug monitoring has lower utility for gabapentin, pregabalin, and vigabatrin. Measurement of salivary drug concentrations has potential utility for therapeutic drug monitoring of lamotrigine, levetiracetam, and topiramate. Therapeutic drug monitoring of the new antiepileptic drugs will be discussed in managing patients with epilepsy.

Clinical utility of free drug monitoring.

Most drugs are bound to serum proteins to a various degree. Only unbound or free drug is pharmacologically active. Usually total drug is measured for therapeutic monitoring because there is equilibrium between bound and free drugs, and concentration of free drug can be predicted from total drug concentration. However, under certain conditions this equilibrium is disturbed and the measured free drug concentration can be significantly higher than expected from total drug concentrations, especially for strongly protein-bound drugs. In such case a patient may experience drug toxicity even if the total drug concentration is within the therapeutic range. Conditions like uremia, liver disease and hypoalbuminemia can lead to significant increases in free drug concentration. Therefore, monitoring free phenytoin and free valproic acid is recommended in these patients. Drug-drug interactions can also lead to a disproportionate increase in free drug concentration. One strongly protein-bound drug can significantly displace another strongly protein-bound drug if both drugs share the same binding site. Several over-the-counter pain medications such as salicylate, naproxen, and ibuprofen can cause significant displacement of both phenytoin and valproic acid from albumin binding site. Interestingly, such interactions are absent in uremic patients. Elderly patients may have increased free phenytoin or valproic acid due to hypoalbuminemia. Elevated free phenytoin concentrations have also been reported in patients with AIDS. Although digoxin is 25% bound to protein, monitoring free digoxin is useful in patients with elevated endogenous digoxin-like immunoreactive substances or in patients overdosed with digoxin and being treated with digibind. Monitoring free digoxin can also eliminate interference of Chinese medicines Chan Su and Danshen in serum digoxin measurement by certain immunoassays. However, free drug monitoring is not a routine procedure in clinical laboratories due to technical difficulties and lack of established reference ranges for free drugs.

Serum free 5-methoxypsoralen fraction in health and psoriasis: relationship with human serum albumin concentration.

Human serum albumin is known to be the main carrier of 5-methoxypsoralen (5-MOP) in serum. As hypoalbuminaemia may occur in psoriasis with inflammatory syndrome, variability of the free 5-MOP fraction in serum can be expected. The free 5-MOP fraction was determined by equilibrium dialysis in serum samples obtained from 18 psoriatic patients and 18 control subjects. The median free 5-MOP fraction was not significantly different in the psoriatic group (fu = 4.75%) than in the control group (fu = 5%). However, there was a significantly larger variability of the free fraction in the psoriatic group (2.7 to 8.6%) than in the healthy group (3.2 to 6.8%) (p = 0.002). The binding index of 5-MOP (ratio of bound to free concentrations) was correlated with human serum albumin level (r = 0.784). This work confirms that the 5-MOP fraction in human serum is principally serum albumin dependent, as has been described with in vitro models. Free drug monitoring of 5-MOP is discussed.

Determination of Free Valproic Acid

Valproic Acid (VPA) is an important drug for the treatment of several types of seizures because it has a wide spectrum of activity. Since VPA has an unusual nonlinear binding characteristic and a wide interindividual variation, monitoring of its free concentration can be helpful in patient management. The determination of unbound VPA is more difficult because an extra sample preparation step is needed and the concentration of free VPA is low. Free drug monitoring can assume a more important role if there is a refinement in the technology. A high-performance liquid chromatography (HPLC) method with isocratic elution has been established for the analysis of the 4-bromomethyl-7-methoxycoumarin (BrMMC) derivative of free VPA. This method has a better sensitivity, linearity, and precision than enzyme immunoassay (EIA). Ultrafiltration with the Centrifree system was evaluated for the sample preparation. The influence of centrifuge times, relative centrifugal forces, and the starting sample amounts on the final results of the ultrafiltration were investigated. There was a satisfactory correlation between the free VPA levels determined by the HPLC method and the concentrations obtained by EIA. The total and free VPA were determined on 100 samples from 36 patients. The total VPA levels were in a range of 25 to 208 micrograms/ml, free VPA concentrations ranged from 1.92 to 55.75 micrograms/ml with the free fractions from 7 to 37%.

Evaluation of Microultrafiltration Devices for Binding Study Using a Small Sample Volume.

The free fraction of drugs was measured with microultrafiltration devices: UltrafreeC3LGC (UFC; Nihon Millipore Ltd., Tokyo, Japan), Centricut W-10 (CCW; Kurabo Industries Ltd., Osaka, Japan), and MPS-1 (Amicon Corp., Danvers, MA). The drugs tested were phenytoin, carbamazepine, and valproic acid. A 200μl sample volume was used. For two of three drugs, the free fraction by CCW was significantly low, and those by UFC did not show constant values. The free fraction by MPS-1 using a small sample volume (200μl) tended to show high values. The variable values of free fraction of drugs obtained with several devices may be caused by protein leakage, adsorption to the device, etc. The accurate free fraction corrected for protein leakage and adsorption to the device was in good agreement in all devices. The results suggest that physicochemical properties of each drug should be noted when a new device is used to examine protein binding of drugs for routine therapeutic free drug monitoring, especially with a small sample volume. In addition, if needed, it is suggested that the free fraction obtained should be corrected for protein leakage and the adsorption to the device.

Comparative Evaluation of Microultrafiltration Devices for Determination of Protein Binding

The free fraction of drugs was determined comparatively with new microultrafiltration devices, Molcut II (Catalogue No. SJGC type) (MLII) and (Catalogue No. PLGC type) (NML; a device derived by changing the membrane component of MLII), which were developed by Nihon Millipore Ltd., Tokyo, Japan. The results obtained with the device are compared with those of MPS-1 (MPS), which is commercially available from Amicon Co., Danvers, MA, U.S.A. Drugs tested are phenytoin, carbamazepine, valproic acid, theophylline, phenobarbital, and gliclazide. In five of six drugs the free fraction by MLII shows lower values, although that by NML is in good agreement with MPS. The lower values of free fraction of drugs obtained with MLII may be caused by adsorption to the device. The adsorption increase is relatively correlated to increasing lipophilicity of drugs. The results suggest that physicochemical properties of each drug, especially hydrophobicity, should be taken into account, when a new device is used to examine protein binding of drugs. The NML, which is less expensive and is convenient because no special instrument is needed, is a reliable and satisfactory device for routine therapeutic free drug monitoring.

Free Drug Monitoring

The principles, technique of measurement, and clinical significance of free drugs are reviewed. Albumin, alpha-1-acid glycoprotein, and lipoprotein are the main drug-binding proteins in blood. Variations of the concentrations of these proteins owing to disease would result in changes in the free drug levels. These levels may be determined by equilibrium dialysis, ultrafiltration, and other techniques.

Free Drug Monitoring by Liquid Chromatography and Implications for Therapeutic Drug Monitoring

Abstract Free (non-protein bound) drug monitoring is very complex but new advances in separation of protein bound from free drug by ultrafiltration can facilitate free concentration therapeutic drug monitoring. Free drug can be separated from protein-bound drug by means of ultrafiltration in which the free drug passes through the membrane filter while the protein bound drug is retained. The ultrafiltrate is then subjected to chromatographic analysis (HPLC). Limitations include a sufficient sample for these highly protein bound drugs and detector sensitivity at the low free drug concentrations. Free drug fractions that have been successfully analyzed by HPLC include disopyramide, phenytoin, carbamazepine, and propranolol. HPLC offers important advantages over immunoassays, since chromatographic methods can also measure the amount of metabolite present. The detection of metabolites can be important, e.g. N-desisopropyl disopyramide which displaces disopyramide from its binding sites on alpha-one acid glycop...

Utility of free level monitoring of antiepileptic drugs.

Criteria that were developed for monitoring free (unbound) rather than total (free plus bound) concentrations of antiepileptic drugs include extensive and variable binding to plasma proteins. Phenytoin and valproic acid belong to this category. It is shown that free drug concentration is independent of total drug concentration, whereas total drug concentration depends on free concentration and free fraction. Because antiepileptic drugs are predominantly bound to albumin, free fraction will increase in the presence of hypoalbuminemia (hepatic and renal disease, burns, and pregnancy). Free fraction also increases because of saturable binding (valproic acid) and competitive binding (valproic acid displacing phenytoin). There is suggestive evidence that side effects may be more closely related to the free, rather than to the total, plasma concentration of phenytoin. The clinical evidence that side effects or therapeutic effects are better correlated to the free, rather than the total, concentration of valproic acid or carbamazepine is not yet convincing. Knowledge of the free concentration improves our understanding of therapeutic and toxic effects of low total plasma concentrations. Further clinical trials are necessary for definitive assessment of the clinical relevance for free drug monitoring of valproic acid, carbamazepine, and phenytoin in the management of epileptic patients.