Free Fraction Of Phenytoin Research Articles

Predicting Unbound Phenytoin Concentrations: Effects of Albumin Concentration and Kidney Dysfunction.

Several methods are available to predict unbound (free) phenytoin concentrations in patients with hypoalbuminemia; however, predictive methods have not been evaluated in patients with concurrent hypoalbuminemia and kidney dysfunction or in patients with mild to moderate (estimated glomerular filtration rate [eGFR] 30-90 ml/min/1.73 m2 ) kidney dysfunction alone. Thus the objective was to evaluate the accuracy and precision of predictive methods to estimate free phenytoin concentrations in patients with varying albumin concentrations and/or kidney dysfunction. Retrospective chart review. Large academic medical center. A total of 344 patients with free and total phenytoin, albumin, and serum creatinine concentrations obtained between November 2012 and May 2017. Free phenytoin concentrations were estimated in patients without kidney dysfunction using the Winter-Tozer, Anderson, Kane, and Cheng equations. For the analysis in patients with eGFR lower than 90ml/min/1.73m2 , free phenytoin concentrations were estimated using the Shiner-Tozer derivation with adjusted affinity coefficients (C=0.15, 0.20, 0.25, and 0.30). For both analyses, accuracy of predictive methods was evaluated by P20, the proportion of estimations within 20% of the measured free phenytoin concentration. In 158 patients with normal kidney function/normal albumin concentrations, 73 with normal kidney function/hypoalbuminemia, or 47 with mild kidney dysfunction/normal albumin concentrations, the Anderson method had the highest accuracy (86%, 82%, and 92%, respectively) and highest precision compared with the other methods. In 47 patients with normal albumin concentrations and mild kidney dysfunction or 13 with moderate kidney dysfunction, the free fraction was unchanged, and total phenytoin concentrations accurately reflected free concentrations. In 17 patients with hypoalbuminemia and mild or 17 with moderate kidney dysfunction, the Winter-Tozer (67% and 50%, respectively) and the Anderson (56% and 67%, respectively) methods had the highest accuracy compared with other methods with significantly lower accuracy compared with patients with normal kidney function. In the 14 patients with severe kidney dysfunction and hypoalbuminemia, none of the coefficients had a P20 accuracy greater than 45%. In patients with normal albumin concentrations, with or without mild or moderate kidney dysfunction and not receiving a protein-binding displacer, the free fraction of phenytoin is unchanged, and it is not necessary to measure a free phenytoin concentration. Free phenytoin concentrations should be measured directly in patients with hypoalbuminemia and kidney dysfunction.

Influences of Artificial Heart-Lung Machine Operation on the Binding Sites of Albumin: Possibility of an Effective Administration Plan

The purpose of this study is to investigate the influence of changes in the human serum albumin (HSA) and free fatty acid (FFA) on alteration of the binding abilities of sites I and II after the operation of an artificial heart-lung machine. The binding abilities of phenytoin (site I) and diazepam (site II) to patients' sera collected before and after the operation of an artificial heart-lung machine and pseudopatient serum samples were examined by ultra-filtration. The binding ability of site I markedly decreased after the operation of an artificial heart-lung machine in all patients, and the binding ability of site II unexpectedly increased in some patients. The variation in pseudopatient serum was similar to that in the patient sera. The difference in the binding ability between sites I and II was due to that the fact that the binding ability of site I is more strongly influenced by HSA level reduction than by [FFA]/[HSA] reduction, whereas the binding ability of site II is more strongly influenced by [FFA]/[HSA] reduction than by HSA level reduction in some patients. Therefore, it may be possible to predict the binding ability of site I by monitoring the HSA level without directly monitoring the free phenytoin fraction (%).

An Interaction Between Aspirin and Valproate: The Relevance of Plasma Protein Displacement Drug-Drug Interactions

An Interaction Between Aspirin and Valproate: The Relevance of Plasma Protein Displacement Drug-Drug Interactions

Analysis of Free Drug Fractions Using Near-Infrared Fluorescent Labels and an Ultrafast Immunoextraction/Displacement Assay

A chromatographic method was developed for measuring free drug fractions based on the use of an ultrafast immunoextraction/displacement assay (UFIDA) with near-infrared (NIR) fluorescent labels. This approach was evaluated by using it to determine the free fraction of phenytoin in serum or samples containing the binding protein human serum albumin (HSA). Items considered in the design of this method included the dissociation rate of HSA-bound phenytoin, the rate of capture of free phenytoin by immunoextraction microcolumns, the behavior of NIR fluorescent labels in a displacement format, and the overall response and stability of the resulting assay. In the final UFIDA method, the free fraction of phenytoin was extracted in approximately 100 ms by a microcolumn containing a small layer of anti-phenytoin antibodies. This gave a displacement peak for a NIR-fluorescent-labeled analogue of phenytoin that appeared within 2-3 min of sample injection, creating a signal proportional to the amount of free phenytoin in the sample. The UFIDA method provided results within 1-5% of those determined by ultrafiltration for reference samples. The lower limit of detection was 570 pM, and the linear range extended up to 10 microM. This approach is not limited to phenytoin but can be adapted for other analytes through the use of appropriate antibodies and labeled analogues.

Interactions of Serum Albumin, Valproic Acid and Carbamazepine with the Pharmacokinetics of Phenytoin in Cancer Patients

Phenytoin dosing is critical in cancer patients as to decreased absorption secondary to chemotherapy-induced gastrointestinal toxicity, increased phenytoin metabolism in the liver secondary to chemotherapy, extreme patient profile that falls outside the predicted pharmacokinetic population, frequent hypoalbuminaemia and polydrug treatment. A retrospective study to assess the variability of free phenytoin and the free fraction of phenytoin, as well as the influence of comedication on these parameters was performed in cancer patients by using a population approach. Two hundred fifty-eight data pairs of total phenytoin and free phenytoin were analysed from 155 cancer patients on stable phenytoin using non-linear mixed-effect modeling (NONMEM). Total and free phenytoin were determined using a fluorescence polarization immunoassay. An extensive model building procedure was subsequently used for covariate testing on the free fraction of phenytoin. Mean total phenytoin concentration was 11.7 mg/l, free phenytoin 1.25 mg/l and phenytoin free fraction 0.107. Free phenytoin was <1 mg/l on 132 occasions (51.2%) and >2 mg/l on 37 occasions (14.3%). Total and free phenytoin were significantly correlated (r(S)=0.827, P<0.01). The free fraction of phenytoin was independent of time after drug intake. Serum albumin concentrations and comedication with valproic acid or carbamazepine were identified by NONMEM as significant determinants of phenytoin free fraction. Co-medication with valproic acid and carbamazepine led to a 52.5% and 38.5% increase of the free fraction of phenytoin, respectively, and a 10 g/l decrease of serum albumin to a 15.1% increase of the free fraction of phenytoin. Phenytoin pharmacokinetics could reliably be estimated from oral doses and steady-state concentrations of protein-bound and free phenytoin. The variability in the free fraction of phenytoin could partly be explained by the influence of albumin concentrations and antiepileptic comedication. Significant alterations of the free fraction of phenytoin and free phenytoin by co-administration of valproic acid or carbamazepine suggest therapeutic drug monitoring of free phenytoin to be of potential benefit in cancer patients.

Clinical evaluation of plasma free phenytoin measurement and factors influencing its protein binding

The relationship between free phenytoin concentrations and clinical responses, and the factors influencing protein binding of phenytoin were investigated. A total of 119 plasma samples from 70 patients treated orally with phenytoin were analysed. The mean free phenytoin concentration was significantly higher in the patients who received phenytoin monotherapy and were classified as having a complete response (1.25 +/- 1.09 microg/ml) than that in the partial response group (0.59 +/- 0.07 microg/ml), whereas the mean total concentrations were not significantly different between the two groups. Samples were divided into three groups based on the free fraction of phenytoin, i.e. low, <5%; medium, 5%-10%; high, > 10%. The mean age (55.3 +/- 10.9 years) was significantly higher in the high group than in the low (42.7 +/- 21.2 years) and medium (42.8 +/- 16.0 years) groups. The mean creatinine clearance (CLcr) (55.3 +/- 10.9 ml/min) and serum albumin concentration (3.30 +/- 1.25 g/dl) were significantly lower in the high group than the low (88.3 +/- 29.0 ml/min and 4.08 +/- 0.50 g/dl, respectively) and medium (95.0 +/- 32.8 ml/min and 3.95 +/- 0.92 g/dl, respectively) groups. These results suggest that the free phenytoin concentration, rather than the total concentration, is more useful for monitoring antiepileptic effects in patients receiving phenytoin monotherapy. It was also found that the free phenytoin fraction was significantly influenced by aging, CLcr and serum albumin levels.

Phenytoin intoxication in critically ill patients

Phenytoin intoxication can result in major and possibly life-threatening disorders. Furthermore, the hepatic clearance can become saturated, thus, shifting the elimination from first- to zero-order kinetics. This results in a slow elimination of the compound in case of intoxication. The treatment modalities for phenytoin overdose are limited. Taking into account the high level of protein binding, the molecule is not easily eliminated from the body by means of extracorporal epuration. Although reports exist on the use of MARS (molecular adsorbents recirculating system) dialysis, peritoneal dialysis, and standard dialysis for the elimination, in practice, hemoperfusion, is the most often applied technique. The authors report the case of a hypoalbuminemic patient with severe neurologic signs of phenytoin intoxication (total concentration moderately elevated, free fraction high). A combination of high-flux dialysis and hemoperfusion resulted in a considerable extraction of the drug, accelerating the natural clearance from the body and ameliorating clinical signs of intoxication. In selected patients (with a high free fraction of phenytoin), high-flux dialysis may be a valuable alternative or adjuvant to hemoperfusion.

Non-invasive monitoring of phenytoin by reverse iontophoresis.

Transdermal iontophoresis offers a non-invasive sampling method for therapeutic drug monitoring. This study examined whether iontophoretic extraction (a) is concentration dependent, (b) reflects the subdermal level of unbound drug, (c) follows protein binding changes, and (d) becomes truly non-invasive when a co-extracted compound is used as an internal standard for calibration. Iontophoresis was conducted in vitro using dermatomed pig-ear skin. The subdermal solution was a buffer containing phenytoin at therapeutic concentrations, an internal standard at fixed level, human albumin and/or valproic acid. The ionized form of phenytoin was recovered at the anode by electro-migration, while the neutral form was extracted to the cathode by electroosmosis. A satisfactory correlation between the reverse iontophoretic extracted amount of phenytoin and the subdermal concentration was observed. Iontophoresis extracted only the free fraction of phenytoin. At steady state, reverse iontophoresis monitored changes in free drug concentration provoked in the subdermal compartment. Acetate was introduced at a fixed concentration into the subdermal compartment to act as an 'internal standard'. Subsequently, acetate and the ionized form of phenytoin were co-extracted to the anode. The ratio of the extracted amounts was proportional to the subdermal concentration ratio demonstrating a means by which the method may become truly non-invasive.

Synergistic Effect of Valproate Coadministration and Hypoalbuminemia on the Serum-Free Phenytoin Concentration in Patients With Severe Motor and Intellectual Disabilities

We investigated whether a combination of risk factors affects the free phenytoin (PHT) fraction by multiple regression analyses in 30 patients with severe motor and intellectual disabilities (SMID) with epilepsy. The risk factors analyzed were gender, age, total PHT concentration, albumin concentration, aspartate aminotransferase, alanin aminotransferase, serum creatinine, blood urea nitrogen, and antiepileptic drug concentrations. Serum levels of total and free PHT were measured by fluorescence polarization immunoassay. Free PHT fractions were between 7.2% and 17.3% (average 10.9%). Two factors, hypoalbuminemia and valproate (VPA) coadministratation with PHT, increased free PHT fraction, and a combination of these two markedly increased free PHT fraction. Patients with these double risk factors have a high risk of exceeding the therapeutic range of serum-free PHT concentration even if their total PHT concentration does not. Therefore, we should monitor free PHT concentration, especially in SMID patients with epilepsy, because they may have hypoalbuminemia and are treated with antiepileptic drug polytherapy and, moreover, cannot report adverse effects of the drugs.

Heparin-induced Release of Protein-bound Solutes during Hemodialysis Is an in Vitro Artifact

Several studies have pointed to a release of drugs or protein-bound solutes from their binding sites during heparinization. The effect is attributed to the metabolism of triglycerides to free fatty acids (FFAs), which compete with drugs for protein binding sites. This study evaluated the impact of intradialytic heparin on the free concentration of the uremic toxin p-cresol and on FFAS: Blood samples from hemodialysis (HD) patients, before and during HD, were collected with selected anticoagulation strategies. We assessed the effects of standing time, temperature, pH, and the addition of a lipase inhibitor, tetrahydrolipstatin (THL) to blood samples on the free p-cresol concentration. p-Cresol was analyzed by HPLC with fluorescence detection. We measured FFAs by gas chromatography, and the free fractions of added valproic acid and phenytoin were evaluated by fluorescence polarization immunoassay. In blood samples (n = 22) not submitted to a specific treatment, free p-cresol increased from 9.9 +/- 5.1 to 31.9 +/- 22.3 micromol/L after 30 min of heparin HD (P < 0.001) and correlated significantly with FFAs (r = 0.80; P = 0.002; n = 12). There was no increase in free p-cresol during heparin-free HD (n = 6) and trisodium citrate HD (n = 9). In addition, p-cresol in ultrafiltrates (n = 3) did not correspond to the free p-cresol in heparinized blood, suggesting that the increase in free p-cresol was artifactual. The release of p-cresol in the test tube was enhanced by standing time (n = 6), sample temperature (n = 6), and alkaline pH (n = 6). Inhibition of lipase activity with THL prevented the increase of FFAs (n = 6) and the release of free p-cresol during HD (n = 22). These results were corroborated by the study of the free fraction of valproic acid (n = 6) and phenytoin (n = 6). The free concentrations of protein-bound solutes in plasma of heparinized patients are influenced by external factors that alter the lipase activity in the test tube. The free fraction does not increase during HD when lipase activity is neutralized at the time of blood sampling, so that previously reported increases are probably artifacts.

In vitro protein binding studies with BMS-204352: lack of protein binding displacement interaction in human serum.

BMS-204352, a maxi-K channel opener, is currently under development for the treatment of stroke. Protein binding of BMS-204352 was determined in sera from several species, namely, rat, monkey, dog, and human. Data indicated that the compound was shown to be highly protein bound in serum from all species (ca. 99.6%). In order to test for the potential for drug-drug interactions and competitive displacement of BMS-204352 by diazepam, phenytoin, propranolol, and warfarin, in vitro experiments were performed using spiked human serum and ex vivo human plasma samples. Protein binding was determined using equilibrium dialysis for 4 h at maximal therapeutic concentrations for each drug alone or in appropriate combination in spiked serum samples. Ex vivo samples from a clinical BMS-204352 study (0, 1, and 24 h) were dialyzed separately after addition of diazepam, phenytoin, propranolol, or warfarin. Drug content in biological matrices was measured for radioactivity using liquid scintillation counting. Results indicated that (1) addition of diazepam, phenytoin, propranolol, or warfarin did not alter the free fraction of BMS-204352; (2) BMS-204352 did not displace diazepam, phenytoin, propranolol, or warfarin from their protein binding sites, and (3) comparison of ex vivo plasma samples after BMS-204352 dosing indicated no impact of BMS-204352 and/or its metabolites on the free fraction of diazepam, phenytoin, propranolol, or warfarin. In conclusion, the potential for a drug-drug interaction due to alterations in protein binding with BMS-204352 is unlikely.

Evidence-based Implementation of Free Phenytoin Therapeutic Drug Monitoring

The majority of laboratories measure total phenytoin concentration for therapeutic drug monitoring. However, there are substantial interindividual variations in free phenytoin concentrations, the pharmacologically active component. We describe the process and data used to implement monitoring of free phenytoin only in an urban medical center. Over a 6-week period, total and free phenytoin concentrations were measured, clinical charts reviewed, and indications for alterations in the percentage of free phenytoin fraction were determined. Of the 189 phenytoin requests from 139 patients, 136 data points were analyzed. Free phenytoin concentrations were 6.8-35.3%, with 50% outside the expected range of 8-12%. Clinical indications likely responsible for variations were hypoalbuminemia, drug interactions, uremia, pregnancy, and age. Overall, 30% of patients demonstrated a discrepancy between therapeutic, subtherapeutic, or supratherapeutic concentrations between free and total phenytoin concentrations. The largest discordance (53%) occurred in the patient group with free phenytoin <8% or >12%. This study supports previous clinical findings that monitoring total phenytoin is not as reliable as free phenytoin as a clinical indicator for therapeutic and nontherapeutic concentrations. Thus, we recommend that therapeutic monitoring should use free phenytoin concentrations only.

The Correlation Study of the Factors Affecting the Phenytoin Free Fraction in Serum

The Correlation Study of the Factors Affecting the Phenytoin Free Fraction in Serum

Pharmacodynamic interaction between phenytoin and sodium valproate changes seizure thresholds and pattern.

1. In this study we used cortical stimulation to assess the effects of phenytoin (PHT), sodium valproate (VPA), and their interaction on total motor seizure and on the constituent elements of the seizure. 2. PHT (40 mg kg(-1)) was administered as an intravenous bolus infusion to animals receiving either a continuous infusion of VPA or saline. VPA plasma concentration was maintained at levels that produced no detectable anticonvulsant effect. 3. Analysis of ictal components (eyes closure, jerk, gasp, forelimb, clonus, and hindlimb tonus) and their durations revealed both qualitative and quantitative differences in drug effects. 4. The anticonvulsant effect is represented by the increase in the duration of the stimulation required to reach a given seizure threshold. PHT significantly increased the duration of the stimulation and of the motor seizure. This increase was greatly enhanced by VPA. In addition, ictal component analysis revealed that the combination of PHT and VPA causes the reduction of a specific seizure component (JERK). 5. Neither the free fraction of PHT nor the biophase equilibration kinetics changes in the presence of VPA. It is concluded that the synergism may be due to a pharmacodynamic rather than a pharmacokinetic interaction.

Factors Affecting the Free Plasma Fraction of Phenytoin in Patients with Epilepsy

The variability and possible factors modifying the free plasma fraction of phenytoin were investigated in 134 patients with epilepsy undergoing long-term treatment. The total and free plasma concentrations of phenytoin were determined using fluorescent polarisation immunoassay. Concentrations of albumin, bilirubin and creatinine were also obtained. The free plasma concentration was separated by ultrafiltration, at 25 degrees C, using Centrifree((R)) filters. Factors related to the free plasma fraction of phenytoin (free plasma concentration/total plasma concentration) were gender, age, dose, therapeutic regimen, total plasma concentration and the biochemical parameters mentioned. The mean of free plasma fraction was 9.1% with a very high variability (between 3.3 and 37%). No significant relationship was found between the free plasma fraction and dose, age, gender, total plasma concentration or the biochemical data. The only variable with a significant effect (p < 0.05) on the free plasma fraction was polytherapy with valproic acid. The variability in the free plasma fraction of phenytoin was high in epileptic patients, and was poorly related to the clinical or analytical variables studied. In the absence of pathologies that modify phenytoin binding (uraemia, hypoalbuminaemia), the only factor predictive of a possible alteration in the binding of phenytoin to plasma proteins was polytherapy with valproic acid.

Phenytoin free fraction determination: comparison of an improved direct serum injection high-performance liquid chromatographic method to ultrafiltration coupled with fluorescence polarization immunoassay

Recent developments in restricted-access media (RAM) liquid chromatography make the simultaneous determination of total and free phenytoin concentrations possible by direct injection of drug-containing serum samples. A comparison of phenytoin free fraction determination by ultrafiltration coupled with fluorescence polarization immunoassay (TDX) to an improved direct injection RAM-HPLC method is presented. Our improved method differs from those previously reported with regard to column type, mobile-phase composition, and column temperature. Replicate samples analyzed by each method yielded similar values for serum phenytoin free fraction.

A Rational Basis for the Measurement of Free Phenytoin Concentration in Critically I11 Trauma Patients

Phenytoin binding to serum proteins and factors influencing protein binding were investigated in 38 critically ill trauma patients. In 24% of these patients, the free fraction of phenytoin was < or = 10%, whereas in 76%, the free phenytoin fraction was increased > 10%--up to 24%. Nonantiepileptic comedication, sex, or age had no influence on phenytoin binding in any of the 38 patients. Elevated free phenytoin fraction was found in those with hypoalbuminemia and hepatic and renal impairments. In these patients, the free phenytoin fraction should be measured routinely.

Fast Atom Bombardment Mass Spectrometric Determination of the Molecular Weight Range of Uremic Compounds that Displace Phenytoin from Protein Binding: Absence of Midmolecular Uremic Toxins

Uremic compounds are known to displace phenytoin from protein binding, resulting in a higher concentration of the pharmacologically active free fraction of phenytoin. The true chemical identities and molecular weight range of all these compounds are still unknown. We demonstrated that indoxyl sulfate and hippuric acid, which are found at high concentrations in uremic patients, can only partially explain the elevated free phenytoin concentration. Other known uremic compounds, guanidine, methylguanidine, and guanidinosuccinic acid, do not displace phenytoin from the protein-binding sites. Uremic compounds from sera of patients on maintenance hemodialysis were removed using activated charcoal. These compounds were then backextracted from activated charcoal using methanol and analyzed by fast atom bombardment mass spectroscopy. Mass spectra of uremic sera showed no peak over m/z 450, indicating that midmolecular uremic toxins are not involved in displacing phenytoin from protein binding. We also observed additional peaks in the mass spectrum of uremic compounds when compared with the normal serum extract, indicating the presence of several endogenous compounds in uremic sera, as expected.

Clinical Response in Epilepsy in Relation to Total and Free Serum Levels of Phenytoin

The relationships between total and free serum concentrations of phenytoin and the clinical control of seizures were investigated retrospectively in 114 patients. Total phenytoin levels were measured by enzyme-modified immunoassay (EMIT), and the free fraction by ultrafiltration at 37 degrees C using 14C-labelled phenytoin as a tracer. The median free fraction in 188 serum samples was 13.7% (range 8.9-27.0%). The free fraction was greater than 18% in 34 (18.1%) of the serum samples. In all but 5 samples, a likely reason for the elevated free fraction could be determined. The identifiable reasons were commonly hypoalbuminaemia and the presence of liver or renal disease. There was a significant negative correlation between serum albumin level and free fraction of phenytoin (n = 90, r = -0.68, p less than 0.001). The free phenytoin concentration was strongly correlated with the total phenytoin concentration in serum (n = 188, r = 0.94, p less than 0.001). The total phenytoin concentration provided as good an indication of clinical response as the free concentration in 91 patients (85.8% of the patients for whom response could be reliably determined). In the other 15 (14.2%) patients, free phenytoin concentrations were better related to clinical effect. These patients generally had significant reductions in the serum protein binding of phenytoin. The relationship between phenytoin toxicity and free serum concentrations was particularly strong--in 14 patients with toxicity, the total serum concentration of phenytoin was greater than 80 mumol/L in only 42.9% of cases, while the free phenytoin concentration was greater than 8 mumol/L in 85.7% of the cases (p less than 0.05 by chi-square test).(ABSTRACT TRUNCATED AT 250 WORDS)

Nomogram for the Prediction of Unbound Phenytoin Concentrations in Patients on a Combined Treatment of Phenytoin and Valproic Acid

The interpretation of the total phenytoin (PT) concentration can be problematic if valproic acid (VPA) is given as a comedication, because VPA displaces PT from the protein binding sites and can increase the free fraction of PT. In order to estimate the free or unbound PT concentration (PTf) from the total PT concentration (PTt) and VPA concentration, a nomogram was constructed and evaluated. Data of 84 patients on combined therapy with PT and VPA were used in drawing up the regression equation (PTf = 0.0792.PTt + 0.000636.PTt.VPA) from which the nomogram was constructed. The predictions were checked using another 33 patients whose serum concentrations were determined in the morning and in addition several times during the day. The results show that using this method the PTf concentrations can be accurately estimated from the PTt and VPA concentrations.