A rapid and sensitive liquid chromatography/tandem mass spectrometry assay for simultaneous quantitation of disopyramide and its major metabolite, mono-isopropyl-disopyramide, in rat plasma and its application to a pharmacokinetic study.

Disopyramide as an antiarrhythmic agent has been used for treating ventricular tachycardia and metabolized into its major metabolite, mono-isopropyl-disopyramide, by CYP3A4. We developed a novel, selective, highly sensitive, accurate, rapid method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of disopyramide and mono-isopropyl-disopyramide in rat plasma. This study is the first report for the assay validation using LC-MS/MS in biological fluids after simple protein-precipitation method. The most sensitive signals by multiple reaction monitoring (MRM) showed at m/z 340.2 → 239.2 and 298.2 → 239.2 with same fragment ion for disopyramide and mono-isopropyl-disopyramide, respectively. The lower limit of quantification (LLOQ) was determined at 2 ng/mL for both analytes and the linear concentration ranges were found to be 2-2000 ng/mL for disopyramide and 2-1000 ng/mL for mono-isopropyl-disopyramide. Finally, this assay was successfully applied to pharmacokinetic analysis of disopyramide and mono-isopropyl-disopyramide after oral and intravenous administration of disopyramide.

Monitoring disopyramide, lidocaine, and quinidine by micellar liquid chromatography.

A micellar liquid chromatography (MLC) method using a C18 column was developed to determine three antiarrhythmic drugs--disopyramide, lidocaine, and quinidine--that are most usually monitored in serum samples. After the application of an interpretative strategy for optimization of sodium dodecyl sulfate (SDS) and modifier concentrations in order to ensure the minimum analysis time, maximum sensitivity, and good resolution, the optimum chromatographic conditions for the determination of the three antiarrhythmics were flow rate, 1 mL/min; injection volume, 20 microL; separation temperature, 25 degrees C; mobile phase, 150 mmol/L SDS-7% (v/v) butanol-phosphate buffer, 10 mmol/L, pH 7-0.9% (w/v) NaCl; and detection at 214 nm. The calibration curves for the drugs were linear (r2 > 0.999). The intraday and interday precisions were lower than 3.9% (CV). Recoveries were 100 +/- 0.6% when the method was applied to both serum samples spiked with the antiarrhythmics (n = 10) and real serum samples. In all cases, the results were similar to those obtained using the reference method (fluorescence polarization immunoassay) usually used in the Spanish hospital. The proposed method is useful for hospital monitoring of the antiarrhythmics by direct injection into the chromatograph.

Effects of serum concentrations of disopyramide and its metabolite mono-N-dealkyldisopyramide on the anticholinergic side effects associated with disopyramide.

The aim of this study was to evaluate the relationship between the anticholinergic side effects associated with disopyramide (DP) and serum DP or mono-N-dealkyldisopyramide (MND) concentrations and the safety range of DP or MND for prevention of anticholinergic side effects in 141 inpatients. The serum DP and MND concentrations were determined by high-performance liquid chromatography. No correlation was observed between creatinine clearance (Ccr) and the ratio of the serum concentration to the dose (C/D) of DP, but a significant inverse correlation was observed between Ccr and the C/D of MND. It was observed that the ratio of MND concentration to DP concentration in the group, whose Ccr was below 20 ml/min, was higher than that of the other groups. Although no significant difference was observed in the DP concentration between the patients without (Group 1) or with (Group 2) anticholinergic side effects, significant differences were observed in the MND concentration, Ccr, and the ratio of MND/DP. The DP concentrations of both Groups 1 and 2 were distributed from 0.13 to about 5 microg/ml. On the other hand, although the MND concentrations of Group 1 were below about 1 microg/ml, the MND concentrations of Group 2 were above about 1 microg/ml. These results suggest that not only DP concentration but also MND concentration should be monitored in patients whose renal function is decreased to prevent anticholinergic side effects associated with DP, and that when serum MND concentration was over approximately 1 microg/ml, the dose should be decreased or discontinued.

Age- and gender-related differences in carbohydrate concentrations of alpha1-acid glycoprotein variants and the effects of glycoforms on their drug-binding capacities.

OBJECTIVE: Alpha(1)-acid glycoprotein (AAG) is a major binding protein for neutral and basic drugs because of its great drug affinity. AAG has three main genetic variants--F1, S, and A variants. Several attempts have been made to elucidate the differences in compositions of the carbohydrate moiety and structure-function relationships such as drug-binding differences. However, there have been few reports on age- and gender-related differences in compositions or concentrations of the carbohydrate moiety of AAG variants. The aim of this study was to clarify the age- and gender-related differences in carbohydrate concentrations and in drug-binding capacities of AAG glycoforms. METHODS: The sera used in this study were obtained from 32 healthy subjects (17 men and 15 women, aged 16-84 years). The AAG glycoforms were isolated by hydroxyapatite chromatography. The binding capacity of AAG to disopyramide (DP), which is a basic drug, was determined using the ultrafiltration method. The concentrations of N-acetylneuraminic acid (NeuAc) and monosaccharides in AAG were determined using high-pH anion-exchange chromatography with pulsed-amperometric detection. RESULTS: The mean plasma AAG concentration in the female subjects was significantly lower than that in the male subjects (0.67 +/- 0.12 mg/ml, mean +/- SD, in females, n = 15, versus 0.81 +/- 0.17 mg/ml in males, n = 17, P < 0.05), but no age-related differences were found (0.75 +/- 0.18 mg/ml in young subjects, n = 24, versus 0.77 +/- 0.12 mg/ml in older subjects, n = 8, n.s.). However, the degree of branching of the glycan chain in the female subjects was significantly lower than that in the male subjects (1.61 +/- 0.17 mol/mol, mean +/- SD, in females, n = 15, versus 1.75 +/- 0.23 mol/mol in males, n = 17, P < 0.05). There was a significant inverse relationship between the binding capacity of AAG to DP (Cb/AAG) and the degree of branching of the glycan chain. The binding capacity (Cb/AAG) decreased as the degree of branching in AAG glycans increased. The binding capacity (Cb/AAG) in the female subjects was significantly higher than that in the male subjects (2.79 +/- 0.59 mg/g AAG in females, mean +/- SD, n = 15, versus 2.37 +/- 0.29 mg/g AAG in males, n = 17, P < 0.05). CONCLUSION. The degree of branching of the glycan chain in AAG plays an important role in drug-binding capacity. Gender-related differences in drug-binding capacity (Cb/AAG) may be caused by differences in the ratios of the extent of branching of the glycan chain in AAG.

Effects of disopyramide and mexiletine on the terminal repolarization process of the in situ heart assessed using the halothane-anesthetized in vivo canine model.

This study was designed to assess the effects of typical class I drugs on the terminal repolarization process of the in situ heart, which is a useful marker of the potential of drug-induced long QT syndrome. Disopyramide (0.3 and 3.0 mg/kg per 10 min, n = 6) or mexiletine (0.3 and 3.0 mg/kg per 30s, n = 6) was intravenously administered to halothane-anesthetized beagle dogs under the monitoring of multiple cardiovascular parameters. Antiarrhythmic concentrations were obtained with the high dose of each drug. The low dose of disopyramide or mexiletine hardly affected any of the electrophysiological parameters assessed. The high dose of disopyramide prolonged the monophasic action potential duration (MAP90) and effective refractory period (ERP) to a similar extent, thus displacing the terminal repolarization period backward, which might provide a potential proarrhythmic substrate, particularly at a slow heart rate. On the other hand, the high dose of mexiletine shortened the MAP90, but prolonged the ERP, resulting in the disappearance of the terminal repolarization period, which could prevent premature excitation with its associated conduction slowing. These electrophysiological effects of disopyramide and mexiletine on the terminal repolarization phase may at least in part explain their clinically described antiarrhythmic and proarrhythmic properties.

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in human plasma by capillary electrophoresis.

In this paper, a rapid method for the enantioselective analysis of the antiarrhythmic drug disopyramide and its main metabolite mono-N-dealkyldisopyramide in human plasma by capillary electrophoresis employing the cyclodextrin-modified electrokinetic chromatography mode is described. Sample clean-up was carried out by alkalinization with sodium hydroxide followed by liquid-liquid extraction with toluene. The complete enantioselective analysis was performed within less than 5 min using 20 mmol/L sodium acetate buffer, pH 5.0, containing 0.2% w/v sulfated beta-cyclodextrin as chiral selector. A 40 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 15 kV and at 20 degrees C. The calibration curves were linear over the concentration range of 62.5-1850 ng/mL and 125-1850 ng/mL for each enantiomer of disopyramide and mono-N-dealkyldisopyramide. The mean recoveries for disopyramide and mono-N-dealkyldisopyramide enantiomers were up to 87 and 69%, respectively. All four enantiomers studied could be quantified at three different concentrations (200, 400 and 600 ng/mL) with coefficient of variation and % relative error not higher than 15%. The quantitation limit was 62.5 ng/mL for (+)-(S)-and (-)-(R)-disopyramide and (-)-(R)-mono-N-dealkyldisopyramide and 125 ng/mL for (+)-(S)-mono-N-dealkyldisopyramide, using 1 mL of human plasma.

Enantioselective analysis of disopyramide and mono-N-dealkyldisopyramide in plasma and urine by high-performance liquid chromatography on an amylose-derived chiral stationary phase.

An enantioselective high-performance liquid chromatography method was developed for the simultaneous determination of disopyramide (DP) and mono-N-dealkyldisopyramide (MND) enantiomers in plasma and urine. The drugs were extracted from plasma samples by liquid-liquid extraction with dichloromethane after protein precipitation with trichloroacetic acid; the urine samples were processed by liquid-liquid extraction with dichloromethane. The enantiomers were resolved on a Chiralpak AD column using hexane-ethanol (91:9, v/v) plus 0.1% diethylamine as the mobile phase and monitored at 270 nm. Under these conditions the enantiomeric fractions of the drug and of its metabolite were analyzed within 20 min. The extraction procedure was efficient in removing endogenous interferents and low values for the relative standard deviations were demonstrated for both within-day and between-day assays. The method described in this paper allows the determination of DP and MND enantiomers at plasma levels as low as 12.5 ng/ml and can be used in clinical pharmacokinetic studies.

Single oral administration of pilsicainide versus infusion of disopyramide for termination of paroxysmal atrial fibrillation: a multicenter trial.

A single oral dose of pilsicainide (PLS) is effective in terminating acute-onset atrial fibrillation (AF). However, the effectiveness of this single oral treatment has not been compared with the infusion of other antiarrhythmic drugs. The effects of a single oral dose of PLS on the termination of AF were compared with an infusion of disopyramide (DISO) in a multicenter trial. Seventy-two patients with electrocardiographically confirmed, symptomatic, paroxysmal AF (< 48-hour duration) were randomized to receive either a single 100- to 150-mg dose of PLS versus a 2 mg/kg (maximum dose = 100 mg) infusion of DISO. Successful defibrillation was defined as termination of AF within 2 hours of drug administration. Conversion of AF to sinus rhythm was achieved within 2 hours in 29 of 40 patients (73%) treated with PLS, and in 18 of 32 patients (56%) treated with DISO (NS). The mean time to return of sinus rhythm was 60 +/- 30 minutes in the PLS group versus 23 +/- 18 minutes in the DISO group (P < 0.001). DISO was particularly effective in terminating nocturnal AF, whereas PLS had a stable circadian effect. PLS was significantly more effective than DISO between 6 AM and 12 PM (64% vs 17%, P < 0.05). No adverse effect was observed in either group. In conclusion, a single oral dose of PLS was as effective as an infusion of DISO to restore sinus rhythm in patients with recent-onset AF. PLS consistently terminated AF regardless of its time of onset.

Effect of sodium channel blockers on ST segment, QRS duration, and corrected QT interval in patients with Brugada syndrome.

INTRODUCTION: Brugada syndrome is characterized by an ST segment elevation in leads V1-V3 and a high incidence of ventricular fibrillation (VF). A mutation in a cardiac Na+ channel gene, SCN5A, has been linked to Brugada syndrome, and sodium channel blockers have been shown to be effective in unmasking the syndrome when concealed. The aim of this study was to examine the effects of Na+ channel blockers on ST segment elevation, QRS, corrected QT (QTc) interval, and ventricular arrhythmias in patients with Brugada syndrome. METHODS AND RESULTS: We examined the effects of three different Na+ channel blockers (flecainide, disopyramide, and mexiletine) on the amplitude of the ST segment 20 msec after the end of QRS (ST20), QRS duration, QTc interval measured from 12-lead ECG, and ventricular arrhythmias in 12 Brugada and 10 control patients. Maximum ST20 observed in the V2 or V3 leads under baseline conditions was greater in the Brugada patients than in control patients, whereas QRS duration and maximum QTc interval were no different between the two groups. Flecainide and disopyramide, but not mexiletine, significantly increased maximum ST20 and QRS duration in both groups, although these effects were much more pronounced in the Brugada patients. The increases in ST20 and QRS duration with flecainide were significantly larger than those with disopyramide. An increase of 0.15 mV in ST20 with flecainide separated the two groups without overlap. Ventricular premature complexes developed only with flecainide in Brugada patients (3/12) displaying a marked ST elevation but not widening of QRS. CONCLUSION: Our findings suggest that Na+ channel blockers amplify existing I(Na) and possibly other ion channel defects, with a potency inversely proportional to the rate of dissociation of the drug from the Na+ channel, thus causing a prominent elevation of the ST segment and, in some cases, prolongation of QRS duration in patients with Brugada syndrome.

Impact of plasmapheresis on disopyramide elimination.

The impact of plasmapheresis on the disposition of disopyramide was investigated in a 16-year-old female with systemic lupus erythematosus. Determination of total disopyramide plasma concentrations immediately prior to and following a 4-h plasmapheresis treatment revealed a significant reduction (i.e., 1.77 to 0.7 mg/l or approximately 60%). However, reassessment of the total serum concentration after 1.5 h (i.e., post equilibrium) revealed a rebounding of the value to 1.64 mg/l. Associated with this reduction in total serum levels was a decrease in the protein-bound fraction of disopyramide from 69.5% (pre treatment) to 48.6% (post treatment) that corresponded to a commensurate reduction in the concentration of alpha1-acid glycoprotein (i.e., 119 mg/dl pre treatment to 48.9 mg/dl post treatment). Despite these alterations in disopyramide concentrations, the procedure removed only 2.7% of the disopyramide dose and was not associated with the appearance of a cardiac dysrhythmia.

Torsades de pointes ventricular tachycardia induced by clarithromycin and disopyramide in the presence of hypokalemia.

We report a 76-year-old woman who developed TdP ventricular tachycardia induced by combined use of clarithromycin and disopyramide. She had a history of myocardial infarction 5 years earlier and has taken disopyramide for supraventricular arrhythmias. In addition, she had taken clarithromycin for upper respiratory tract infection. On admission, an ECG showed prolongation of QTc interval to 0.71 seconds and self-terminating TdP occurred several times. Disopyramide was metabolized by the cytochrome enzyme CYP3A4 and clarithromycin competitively inhibits this enzyme, probably resulting in an increase in plasma concentration of disopyramide. We should consider this possibility when prescribing clarithromycin in combination with antiarrhythmic agent disopyramide.

Effects of oral repetitive loading of disopyramide on acute-onset atrial fibrillation with concurrent monitoring of serum drug concentration.

We investigated the efficacy and safety of oral repetitive loading of disopyramide, for the termination of acute-onset (i.e., therapy started within 2 days after the onset of palpitations) atrial fibrillation (AF) in 96 consecutive patients, with concurrent monitoring of the serum concentration of this agent in fifteen of the patients. Outpatients with AF verified by standard electrocardiogram (ECG) were hospitalized and received disopyramide (200 mg) every 4-6 h, with a maximal dose of 800 mg daily, until the termination of AF under ECG monitoring was obtained. Conversion to sinus rhythm occurred within the first day of treatment in 88 patients (92%), on the second day of treatment in six patients (6%), and on the third and fifth days of treatment in the remaining two patients. No major adverse effects, such as hypotension, congestive heart failure, proarrythmic events or systemic embolism were noted. The serum levels of disopyramide evaluated in fifteen of the enrolled patients were found to be maintained within the therapeutic range throughout the treatment period. In spite of the absence of any placebo-controlled group in this study, these findings suggest that repetitive oral loading of disopyramide (200 mg) with an interval of 4-6 h is effective and safe for the termination of acute-onset AF under a stable therapeutic serum drug concentration, hence offering the possibility of self-medication for patients with episodic AF.

Isotachophoretic determination of bisoprolol, clonidine, disopyramide and tolazoline in human fluids.

Separation and determination of bisoprolol, clonidine, disopyramide and tolazoline in control serum and in human urine was investigated by capillary isotachophoresis. The drugs were separated by using the cationic electrolyte system. viz., sodium acetate buffer (pH 4.64) (c1 = 10 mM)-beta-alanine. The compounds were almost totally isolated from serum by solid-phase extraction using a Sep-Pak C18 cartridge. The recovery of compounds varied from 87 to 99%. The linear calibration range was studied to apply the method to real human fluids. The limit of determination of the drugs was 40.0 micrograms/ml serum. The limit of determination by direct sampling for bisoprolol is 3 micrograms/ml urine.

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography.

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(-)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 - 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(-)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2-6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(-)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5-6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.

Mechanism of compound- and species-specific food effects of structurally related antiarrhythmic drugs, disopyramide and bidisomide.

PURPOSE: To determine mechanism of food effects observed with bidisomide but not with the structurally similar drug, disopyramide. METHODS: Food effect studies of bidisomide and disopyramide were conducted with and without a standardized high fat meal in healthy subjects and in the dog. Intestinal metabolism of disopyramide and absorption of the metabolites were examined after oral administration of the drug to the dogs with portal vein canula implanted. Effects of food or a mixture of amino acids on metabolism of [14C]disopyramide were examined after intraportal infusion of the drug with and without high fat meal and after drug infusion into portal vein with the amino acid mixture, respectively. RESULTS: The systemic availability of bidisomide was markedly reduced with food in humans, whereas the systemic availability of disopyramide did not change notably. In the dog, the systemic availability of bidisomide was also reduced with food. The systemic availability of disopyramide did not change with food. This was due to the fact that reduction in absorption was compensated by reduction of metabolism. There was no evidence for reduction in hepatic and intestinal metabolism with food. CONCLUSIONS: The apparent reduction in disopyramide metabolism with food may be due to an increase in colonal and/or lymphatic absorption. Food effects on the apparent systemic availability of bidisomide and disopyramide in the dog were similar to those in the rat. However, there was substantial species difference in the mechanism of food effects.

Comparison of the effects of cimetidine and ranitidine on the pharmacokinetics of disopyramide in man.

The widely prescribed antiulcer agents cimetidine and ranitidine have the potential to affect the absorption, metabolism or renal excretion of disopyramide. This study investigated the effect of a single oral dose of cimetidine or ranitidine on the pharmacokinetics of disopyramide and mono-N-dealkyldisopyramide in six healthy volunteers. The treatment was conducted in a randomized cross-over design. Serum levels and urinary recoveries of disopyramide and mono-N-dealkyldisopyramide were assayed by HPLC. Cimetidine significantly elevated the maximum plasma concentration of disopyramide, the area under the plasma concentration-time curve and the total amount of disopyramide excreted unchanged in the urine, but the serum profile of mono-N-dealkyldisopyramide was not significantly affected. The effects of ranitidine on the pharmacokinetics of disopyramide and mono-N-dealkyldisopyramide were not significant. The interaction between cimetidine and disopyramide occurred mainly at the site of absorption. The results indicate that cimetidine, but not ranitidine, significantly increased the absorption of orally administered disopyramide.

Fármaco antiarrítmicos / Anti-arrhythimia agents

O diagnóstico correto do tipo de arritmia cardíaca é a primeira etapa na avaliação do paciente. A necessidade da terapêutica antiarrítmica deve ser cuidadosamente avaliada para, em seguida, decidir se a abordagem será farmacológica ou não. A escolha do fármaco antiarrítmico deve ser individualizada, considerando-se a farmacocinética e as interaçöes medicamentosas. A identificação e correção de condiçöes associadas (isquemia miocárdica, disfunção ventricular, distúrbios eletrolíticos) e a avalização periódica da função dos órgãos responsáveis pela metabolização e excreção das drogas são fundamentais para minimizar os efeitos pró-arrítmicos.

Differences between blacks and whites in plasma protein binding of drugs.

OBJECTIVE: Disopyramide and salicylic acid were used as model compounds to characterize racial differences in binding of drugs by alpha 1-acid glycoprotein (AGP) and albumin, respectively. Drug-free plasma was collected from 29 healthy volunteers (15 white, 14 black). Disopyramide and salicylic acid unbound fractions (fu) in plasma were determined by equilibrium dialysis using 14C-disopyramide and 14C-salicylic acid. RESULTS: Disopyramide unbound fractions were significantly higher in blacks than whites (0.131 vs 0.113) as were salicylic acid unbound fractions (0.053 vs 0.048). When unbound fractions were corrected for AGP and albumin concentration, racial differences were no longer present. CONCLUSION: Many drugs which bind to AGP and/or albumin may exhibit racial differences in unbound fractions. However, these differences are likely explained by differences in protein concentrations rather than differences in the number of binding sites on the protein or racial differences in affinity of the protein for drugs.

Disopyramide concentrations in human plasma and saliva: comparison of disopyramide concentrations in saliva and plasma unbound concentrations.

OBJECTIVE: This study was performed to investigate whether it is possible to use saliva instead of blood usually used for therapeutic drug monitoring (TDM) of disopyramide. METHODS: Six healthy male volunteers ingested 200 mg of disopyramide base, and the disopyramide concentrations in saliva and plasma (total and unbound) were determined by the HPLC. RESULTS: Disopyramide concentration-time profiles for the saliva were nearly equal to those for the plasma unbound concentrations. A large variation for absorption time of the drug was observed among the subjects. Disopyramide concentrations (Cs) in saliva did not correlated well with plasma total concentrations (Cp), r = 0.799, but did well with unbound concentrations (Cpu), r = 0.969, for the 3-12 h period on the elimination phase. The mean ratio of disopyramide concentrations in the saliva against the plasma unbound concentrations was almost constant (1.02(0.10), CV = 9.7%) for the period. The pharmacokinetic parameters (tmax, t1/2, AUC, AUMC and MRT values) for disopyramide calculated from the saliva data were nearly equal to those from the unbound data. CONCLUSION: Disopyramide concentrations in saliva correlated well with plasma unbound concentrations on the elimination phase.

Binding characteristics of KNI-272 to plasma proteins, a new potent tripeptide HIV protease inhibitor.

The binding characteristics of KNI-272, a potent and selective human immunodeficiency virus (HIV) protease inhibitor, were evaluated in rat and human plasma, and in solutions of human alpha 1-acid glycoprotein (AAG) and human serum albumin (HSA). The unbound fractions (Fu) of KNI-272 were 12.13 and 2.24% in rat and human plasma, respectively, at the drug concentration of 1.0 microgram mL-1. Although KNI-272 binds to both AAG and HSA, the Fu of KNI-272 in AAG solution was 1.83%, and only one-quarter of that in HSA solution (Fu = 6.78%). Binding displacing agents, such as disopyramide, warfarin, diazepam, and digitoxin, were used to determine the binding site of KNI-272 on these plasma proteins. The Fu of KNI-272 in AAG solution increased 14-fold when disopyramide was added to the AAG solution. In addition, warfarin, diazepam, and digitoxin were added to HSA solution as representative drugs bound to distinct binding sites on HSA, namely sites I, II, and III, respectively. The Fu values of KNI-272 in HSA solution significantly increased when warfarin and diazepam were added. In particular, with the addition of warfarin to HSA solution, the Fu of KNI-272 increased to 16%. The modified Scatchard plots of KNI-272 binding to AAG and HSA both showed biphasic curves, and the KNI-272 binding sites at low concentration range on AAG and HSA disappeared with the addition of disopyramide and warfarin, respectively. Therefore, it is considered that KNI-272 binds to the identical site as disopyramide on AAG and site I on HSA in the low KNI-272 concentration range. By comparing the KNI-272 binding parameters obtained in human plasma and these protein solutions, we can assume that KNI-272 binding at low concentration in human plasma is mainly concerned with the binding on AAG. As KNI-272 concentration in plasma increases, HSA becomes concerned with KNI-272 binding.