INTRODUCTION
A drug in plasma is bound to proteins. This binding, called plasma protein binding, is a reversible and kinetically rapid interac- tion. Unbound drug transfers freely to the target organ, whereas bound drug hardly pass through the blood capillary walls. As a result, unbound drug concentration shows much better correlation with pharmacological activity than the total (bound + unbound) drug concentration. Some important pharmacokinetic properties such as hepatic metabolism rate, renal excretion rate, biomembrane perme- ation rate and steady-state distribution volume are the function of the unbound drug fraction (unbound / total concentration ratio) [1- 3]. Several plasma proteins such as albumin, α i -acid glycoprotein (AGP) and lipoproteins contribute simultaneously to plasma protein binding of one drug. The binding properties of these plasma proteins are different from each other, and their plasma concentrations may vary depending on gender, age and/or disease state of patients. Because of these complex and dynamic characters, it is necessary to investigate the binding property of individual plasma protein for the *Corresponding author: Tel, +81-75-753-4531; fax, +81-75-753-4578; e-mail, akimasas@pharm.kyoto-u.ac.jp comprehensive elucidation of drug distribution in plasma. In case of a racemic drug, the enantioselective plasma protein binding study is inevitable, because usually only one enantiomer shows the pharma- cological activity, and plasma protein binding affinity is often differ- ent between the enantiomers [4,5]. So far, several methodologies have been proposed for the drug - plasma protein binding study [6,7]. Equilibrium dialysis or ultrafil- tration followed by HPLC analysis is the most commonly used to determine unbound drug concentration in plasma protein binding equilibrium. However, these conventional methods are often inapplicable to the analyses of strongly bound drugs because of technical problems such as drug adsorption on the membrane and the leakage of bound drug through membrane. To overcome these problems, we developed high-performance frontal analysis (HPFA) method, a novel binding analysis method using a separation system such as HPLC and capillary electrophoresis. This method has been applied to the highly sensitive analysis of strongly bound drug, enantioselective protein binding study, ultramicro binding study of AGP and lipoproteins [8-29]. This review article deals with the application of HPFA method to the enantioselective plasma protein binding study of semotiadil and levosemotiadil, an enantiomeric pair of drugs under development (chemical structure, see Fig.1) [21-23]. Semotiadil (R-isomer) is a calcium antagonist with non- dihydropyridine structure and shows antihypertensive [30,31] and antianginal activities [32,33].  Levosemotiadil (S-isomer) is an antiarrhythmic drug with sodium and calcium channel blocking action [34-39], as well as potassium blocking activity [40]. The optical purity of each enantiomer, determined by the chiral HPLC system, was more than 99%. Both enantiomers are highly bound to plasma proteins, and the unbound drug concentration could not be determined precisely using any conventional methods.
PRINCIPLE AND FEATURES OF HPFA
1. Principle of HPFA
HPFA method uses a restricted-access type HPLC column [41,42]. This type of columns have the nature to exclude large molecule of plasma protein but to retain a drug of small molecular size mainly in the micropores. Therefore, this type of columns allow a direct injection analysis of plasma samples without deproteiniza- tion. If the injection volume is small, the sample solution is diluted with mobile phase, and the bound drug is released from protein. However, if the sample solution is injected continuously, the release of the bound drug in the column is apparently suppressed, and finally an equilibrium zone is generated near the top of the column. In this zone, two different equilibrium states are established simulta- neously. One is the chromatographic partition equilibrium inside the micropores, and the other is drug-protein binding equilibrium in the interstices of packed particles (outside the micropores). The drug concentration in the stagnant flow of mobile phase in the micropores is equal to the unbound drug concentration in the bulk mobile phase in the interstices. Since the protein concentration is the same with that in the initial sample solution, the unbound drug concentration in the mobile phase is also equal to that in the initial sample solution. That is, the mobile phase in the interstices is replaced by the sample solution. After the end of sample injection, the drug is separated from the protein. During the separation, the drug- protein binding equilibrium is kept constant, because plasma protein binding is a reversible and kinetically rapid process. Then, the unbound drug zone is generated in the column. This drug zone is eluted as a trape- zoidal peak with a plateau region, and the drug concentration in the plateau region is equal to the unbound drug concentration in the initial sample solution. The plateau height and the peak area corre- spond to the unbound drug concentration and total drug concentra- tion, respectively. This is the principle of HPFA method, and unbound drug concentration is determined from the plateau height or by on-line or off-line analysis of the plateau region.
2. Features of HPFA Method
The features of HPFA method, which were described in detail elsewhere [43], can be summarised as follows, 1) HPFA allows a simple binding analysis following direct sample injection, and does not suffer from undesirable drug adsorption onto membrane nor leakage of the bound drug from the membrane, which are often encountered in the conventional ultrafiltration and equilibrium methods. 2) In HPFA, bound drug is not separated from unbound drug, but is transformed into unbound form. As a result, not only unbound drug concentration but also total drug concentration can be determined simultaneously, as in case of carbamazepine binding analyses [9] . 3) HPFA can be easily incorporated into on-line HPLC system. By coupling HPFA column with a chiral HPLC column, the unbound concentration of a racemic drug can be determined enantioselectively. 4) The detection limit can be markedly improved by coupling HPFA column with a preconcentration column. 5) Frontal analysis achieved in capillary electrophoresis format (HPFA/CE) enables the binding assay using very small sample injection volume (ca. 100nL).
PROTEIN BINDING STUDY OF SEMOTIADIL AND
LEVOSEMOTIADIL
1. On-line HPFA/HPLC system
Figure 2 shows the schematic diagram of the on-line HPFA/HPLC system. HPFA column, extraction column and analyt- ical column were connected via four-port and six-port switching valves. The instruments used are as follows: HPLC pumps, LC 6A (Shimadzu, Kyoto, Japan) and Twincle (Jasco, Tokyo, Japan); UV detectors, SPD-2A and SPD-6A, (Shimadzu); injector, Rheodyne Type 8125 equipped with a 5-mL loop; integrated data analyzer, Chromatopac C-R3A and C-R6A (Shimadzu). Table 1 lists the HPLC conditions. Sodium phosphate buffer (pH 7.4, I=0.17) without adding any organic modifier was used as the mobile phase of HPFA so as not to disturb the drug-protein
* The phosphate buffer (pH7.4, I=0.17) contains 13.5mM NaH 2 PO, and 52.2mM Na 2 HPO 4 , and the phosphate buffer (pH6.5, I=0.05) contains 24.0mM NaH,PO, and 8.68mM Na,HPO,. Methanol and acetonitrile, both of HPLC grade, were purchased from Wako (Osaka, Japan).. binding equilibrium. A diol-silica column was used as the HPFA column. This column allows the elution of the hydrophobic drugs under the mild mobile phase condition. HPFA - achiral HPLC system was used when the sample solutions contain only one enantiomer. HPFA - chiral HPLC system was used in the competi- tive binding study where the sample solutions contain both enantiomers.
2. Determination of unbound drug concentrations
The drug - protein mixed solution was directly injected to the HPFA column. The drug was eluted as a zonal peak with a plateau region, and the drug concentration in the plateau region is equal to the unbound drug concentration in the sample solution. Then, a given volume of the plateau region was transferred into the extrac- tion column by switching the 4-port valve ("heart-cut" procedure). The unbound drug was completely trapped on this column. By switching the 6-port valve, the mobile phase for the analytical column was introduced into the extraction column, and the trapped unbound drug was completely transferred to the analytical column. The area of drug peak (retention time, 3.5 min) was measured at 230 nm. The extraction column was washed with distilled water for 1 min before and 30 s after the heart-cut procedure.
3. Optimization of Sample injection volume
Sample injection volume is a crucial factor in HPFA analyses. If the injection volume is insufficient, the original drug - protein Sample solutions, 80μM semotiadil - 100μM HSA mixed solution. binding equilibrium is not reproduced in the column, and the true values of unbound drug concentration cannot be determined. Therefore, it is essential to decide the sufficient injection volume to obtain the plateau region. Figure 3 shows the chromatograms of 80μM semotiadil - 100μM HSA mixed solution which contains the highest unbound drug fraction among the samples used in this binding study. The drug peak height increased with increasing injec- tion volume, and finally reached the maximum level.  When a 5-mL portion of the sample solution was injected, a clear plateau peak appeared. Further increase in the injection volume resulted in expan- sion of the plateau region but did not raise the plateau height. Therefore, the injection volume was decided as 5mL. It is necessary to prevent the diffusion of the sample solution in the injector loop, because otherwise the binding equilibrium may be disturbed and the plateau region may disappear. The "injector- reswitching technique" is useful to overcome this problem. 12-15 The sample solution was injected as follows. The injector loop was loaded with a 6-mL portion of the sample solution and connected to the mobile phase flow (1mL/min) for 5 min. Therefore, the actual injection volume was 5mL. Due to the above-mentioned procedure, the sample input was regarded as an ideal rectangular shape. In the similar way, the actual injection volume for AGP binding study was optimized as 667μL.
4. Estimation of binding parameters
Figure 4 shows the Scatchard plots of HSA binding and human AGP binding, respectively. The binding parameters were estimated
Heart-cut volume, 20 mL. by fitting the experimental data to the following equation
r/Cu = -Kr + nK
where r, Cu, K and n represent the number of moles of bound drug per one mole of HSA, the unbound drug concentration, binding constant and the number of binding site on one HSA molecule, respectively. Both enantiomers are bound to HSA or human AGP at a single binding site. The correlation coefficient of the lines was - 0.97 ~ -0.99, indicating a good agreement of the experimental data with the theoretical equation. The contribution of the binding at non- specific (secondary) binding sites was not observed. The binding parameters and the total binding affinity (nK) are listed in Table 2. It was found that the total binding affinity of AGP Figure 5. Effect of warfarin and diazepam upon unbound fraction of semotiadil and levosemotiadil in HSA solutions. Sample solutions, 10μM semotiadil or levosemotiadil + 100 μM HSA with/without 100μM warfarin or 40μM diazepam (pH 7.4, I=0.17, 37°C). Warfarin (Ca.No. A2250) and diazepam (Ca.No. D0899) were purchased from Sigma. is 110 times for (R)-isomer and 30 times for (S)-isomer stronger than that of HSA. The orientation in chiral discrimination is opposite between HSA and human AGP. Whereas the binding of (S)-isomer to HSA is about three times stronger than that of (R)- isomer, the binding of (R)-isomer to AGP is about 1.2 times stronger than that of (S)-isomer.
5. Identification of the binding site on HSA molecule
The warfarin and diazepam binding sites are the two major binding sites on HSA molecule where many drugs are bound. To identify the binding site of semotiadil and levosemotiadil, the change in the unbound drug fraction caused by the addition of warfarin or diazepam was investigated. Figure 5 compares the unbound fraction of semotiadil or levosemotiadil in 100 μ M HSA solutions in the absence or presence of 100μM warfarin or 40μM diazepam. The unbound concentrations of both enantiomers were not changed by the addition of warfarin. On the other hand, their unbound fractions were increased more than two-fold by the addition of diazepam. This result suggests that both enantiomers are bound at diazepam site.
6. Enantiomer-enantiomer interaction on AGP binding
The present system was applied to investigate whether both enantiomers are bound at a single binding site on AGP molecule. Figure 6 shows the unbound concentrations of (R)- and (S)-isomers in a series of sample solutions containing 25μM AGP, 3, 5 or 7μM (R)-isomer and 5 μ M (S)-isomer. Incorporation of the chiral HPLC column into the on-line HPLC system allowed the determination of unbound concentrations of both enantiomers simultaneously. The lines in Figure 6 show the theoretical values calculated assuming the competitive binding (line 1) and the independent binding (line 2). The observed values are in good agreement with the calculated values based on the competitive binding mode. The agreement indicates that both enantiomers are bound at the same binding site competitively without any kind of allosteric effect.
7. Determination of unbound drug concentrations in human
plasma
The present method was applied to the analysis of human plasma samples. Table 3 shows the unbound drug concentrations in human plasma samples. The total drug concentrations are in their therapeutic levels. The unbound concentration as low as 3nM can be determined with good reproducibility using a common UV detector. This is because the unbound drug in as large as 20mL of plateau region was preconcentrated in the present on-line HPLC system. The bound fractions of both enantiomers are more than 99%. Likewise in the AGP solutions, the unbound concentration of R- isomer in human plasma was lower than that of S-isomer. This means that AGP plays a principal role in the enantioselective plasma protein binding. In conclusion, semotiadil is bound to AGP 1.2 times stronger than levosemotiadil. This enantioselectivity is opposite to that of HSA. Because the affinity of AGP to these enantiomers is 100 or 30 times stronger than HSA, the protein binding in human plasma reflects the enantioselectivity of AGP. Both enantiomers are bound at a single common binding site on AGP molecule competitively. The present study demonstrates the utility of HPFA method in quantitative and enantioselective plasma protein binding study of highly bound drugs.