Studies of Protein Binding to Nonpolar Solutes by Using Zonal Elution

to load: https://cdn.mathjax.org/mathjax/contrib/a11y/accessibility-menu.js ..... This indicated that cis- and trans-clomiphene were sufficiently ...
0 downloads 0 Views 103KB Size
Download PDF
Anal. Chem. 1998, 70, 4602-4609

Studies of Protein Binding to Nonpolar Solutes by Using Zonal Elution and High-Performance Affinity Chromatography: Interactions of cis- and trans-Clomiphene with Human Serum Albumin in the Presence of β-Cyclodextrin David S. Hage* and Arundhati Sengupta

Chemistry Department, University of Nebraska, Lincoln, Nebraska 68588-0304

High-performance affinity chromatography and zonal elution studies were used to examine the binding that takes place between the drug clomiphene and the protein human serum albumin (HSA). Equations were derived to describe the behavior of zonal elution experiments in which a solubilizing agent is present in the mobile phase to aid in the dissolution of a competing agent or injected analyte. These equations were then used to determine the association equilibrium constants for the clomiphene/ HSA system, with β-cyclodextrin being used as a complexation agent to improve the water solubility of cis- and trans-clomiphene without affecting the nature of their binding to HSA. It was found in these studies that both cis- and trans-clomiphene have 1:1 interactions at a common binding region on HSA (association constants at pH 7.4 and 37 °C: cis, 7.5 × 106 M-1; trans, 1.3 × 106 M-1). Further competition experiments between cisor trans-clomiphene and various site-selective probes indicated that the clomiphene-binding region is the same as the proposed tamoxifen site of HSA. The approach and equations used within this report are general ones that can be applied to zonal elution studies of other soluteligand systems in which one or more of the test components have limited solubility in the desired mobile phase.

cases).5-7 In addition, there is growing evidence that immobilized HSA can successfully be used in modeling the behavior of HSA in solution. For example, it has been shown that association constants measured by equilibrium dialysis for soluble HSA with (R)- and (S)-warfarin or L-tryptophan (i.e., solutes that interact with one of the two major binding regions of HSA) are in close agreement with values determined using immobilized HSA columns.8-10 It has also been found that displacement phenomena and allosteric interactions observed for HSA columns are representative of the behavior noted for HSA in solution.10-16 Zonal elution is one technique that has frequently been used to study the binding and competition of drugs and other solutes on immobilized HSA columns.4,17,18 This method is generally performed by injecting a sample of the drug or solute of interest onto an HPAC column (containing HSA or some other immobilized ligand) and in the presence of a fixed concentration of a competing agent in the mobile phase. Analysis of the results is performed by determining how the retention time or capacity factor for the injected solute changes as a function of the competing agent’s concentration. This can be used to provide not only qualitative information on binding and displacement but also quantitative information on the equilibrium constants for these processes.5,10,14-16,19,20 However, this method does require that a sufficient quantity of competing agent can be dissolved in the

Many drugs that enter the blood stream are bound to one or more carrier proteins that help control the transportation, metabolism, and excretion of these drugs.1 Human serum albumin (HSA) is the most abundant protein in human plasma and is an important binding agent for many drugs and endogenous compounds (e.g., fatty acids or bilirubin).2,3 There has been increasing interest in recent years in using immobilized HSA columns and high-performance affinity chromatography (HPAC) to model and predict the protein binding of drugs in the body.4 Some advantages of this approach include its relative precision and speed and its ability to reuse the same ligand preparation for multiple experiments (e.g., up to 500-1000 injections per column in some

(5) Loun, B.; Hage, D. S. J. Chromatogr., B 1995, 665, 303. (6) Loun, B.; Hage, D. S. Anal. Chem. 1996, 68, 1218. (7) Yang, J.; Hage, D. S. J. Chromatogr., B 1996, 725, 273. (8) Loun, B.; Hage, D. S. Anal. Chem. 1994, 66, 3814. (9) Yang, J.; Hage, D. S. J. Chromatogr. 1993, 645, 241. (10) Loun, B.; Hage, D. S. J. Chromatogr. 1992, 579, 225. (11) Domenici, E.; Bertucci, C.; Salcadori, P.; Motellier, S.; Wainer, I. W. Chirality 1992, 2, 263. (12) Domenici, E.; Bertucci, C.; Salvadori, P.; Felix, G.; Cahagne, I.; Motellier, S.; Wainer, I. W. Chromatographia 1990, 29, 170. (13) Domenici, E.; Bertucci, C.; Salcadori, P.; Wainer, I. W. J. Pharm. Sci. 1991, 80, 164. (14) Noctor, T. A. G.; Pham, C. D.; Kaliszan, R.; Wainer, I. W. Mol. Pharmacol. 1992, 42, 506. (15) Noctor, T. A. G.; Wainer, I. W.; Hage, D. S. J. Chromatogr. 1992, 577, 305. (16) Hage, D. S.; Noctor, T. A. G.; Wainer, I. W. J. Chromatogr., A 1995, 693, 23. (17) Sebille, B.; Zini, R.; Madjar, C. V.; Thuaud, N.; Tillement, J. P. J. Chromatogr. 1990, 531, 51. (18) Wainer, I. W. J. Chromatogr., A 1994, 666, 221. (19) Rahim, S.; Aubry, A.-F. J. Pharm. Sci. 1995, 84, 949.

(1) Koch-Weser, J.; Sellers, E. M. New Engl. J. Med., 1976, 294, 311. (2) Kragh-Hansen, U. Pharmacol. Rev. 1981, 33, 17. (3) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153. (4) Hage, D. S.; Tweed, S. A. J. Chromatogr., B 1997, 699, 499.

4602 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

10.1021/ac980734i CCC: $15.00

© 1998 American Chemical Society Published on Web 10/06/1998

concentration of a competing agent (I) (i.e., cis- or transclomiphene) was continuously applied to a column that contained an immobilized ligand (L) (i.e., HSA) while injections of a small amount of analyte (A) were made. If I and A compete for a single site on L, and A binds to no other type of site on the column, then the following reactions can be used to describe the binding events taking place in the column KAL

A + L y\z A-L KIL

I + L y\z I-L

Figure 1. Structures of cis-clomiphene (or zuclomiphene) and transclomiphene (or enclomiphene).

mobile phase (i.e., a physiological buffer) in order to cause an observable shift in analyte retention.4 This requirement can be a problem when dealing with solutes that are only sparingly soluble in aqueous solution. One goal of this present study will be to investigate a way of overcoming this limitation by using cyclodextrins as secondary complexing agents for nonpolar drugs that are to be used as competing agents in zonal elution studies on HPAC columns. The model drug that will be considered in this work is clomiphene, or 1-[p-(β-diethylaminoethoxy)phenyl]-1,2-diphenylchloroethylene. Clomiphene is a synthetic estrogen agonist/ antagonist which has been used for the induction of ovulation in anovulatory women and for the treatment of oligospermia in men. Clomiphene is a relatively nonpolar compound that exists in two isomeric forms, cis- and trans-clomiphene (see Figure 1), which differ in their therapeutic and pharmacological properties. Pharmaceutical preparations of clomiphene consist of roughly a 1:2 mixture of the cis and trans isomers.21,22 Although it is known that clomiphene binds to HSA,21,23 there is no current information available on the interactions between HSA and the separate isomers of this compound. In this study, binding data for the individual isomers will be obtained by using zonal elution and immobilized HSA columns to examine the competition of cis- and trans-clomiphene with themselves or each other, as well as with other compounds that act as markers for specific binding regions on HSA, such as warfarin, L-tryptophan, and tamoxifen.8-10,23 These data should help provide a more complete picture of the interactions of clomiphene with HSA in the body and on how zonal elution HPAC can be used to examine the binding of nonpolar compounds to immobilized proteins. THEORY The binding of cis- and trans-clomiphene to HSA was studied by using the technique of zonal elution.4 In this method, a known (20) Aubry, A.-F.; Markoglou, N.; McGann, A. Comp. Biochem. Physiol. 1995, 112C, 257. (21) Clark, J. H.; Markaverich, B. M. Pharmacol. Ther. 1982, 15, 467. (22) U ¨ rmo ¨s, I.; Benko¨, S. M.; Klebovich, X. J. Chromatogr. 1993, 617, 168. (23) Sjoholm, I. In Drug-Protein Binding; Reidenberg, M. M., Erill, S. Eds.; Praeger Publishers: New York, 1986; Chapter 4.

(1) (2)

where KAL and KIL are the association equilibrium constants for the formation of the complexes A-L and I-L, respectively, between A or I and the immobilized ligand. This study also required the use of β-cyclodextrin as a mobilephase additive to aid in the solubilization of cis- and transclomiphene. If such a solubilizing agent (S) has 1:1 interactions with both A and I, then the following reactions will occur within the mobile phase during the zonal elution study KAS

A + S y\z A-S KIS

I + S y\z I-S

(3) (4)

where KAS and KIS are the association equilibrium constants for the formation of the complexes A-S and I-S, respectively, between A or I and the solubilizing agent. For the given reaction scheme, the following equation (see derivation given in the Appendix) describes the change in retention for A that would be expected as the mobile-phase concentration of either the competing agent or solubilizing agent is varied, with the solubilizing agent being present in a large excess versus both A and I.

1/kA′ )

(1 + KASCS)(1 + KISCS) KALCL (1 + KILCS)

+

(1 + KASCS)(KILCI) KALCL (1 + KILCS)

(5)

In eq 5, the term kA′ is the capacity factor for the injected solute, or kA′ ) (tR/tM - 1), where tR is the measured retention time of the solute and tM is the column void time. The term CL is the total concentration of all active ligand sites in the column, CS is the total concentration of the solubilizing agent, and CI is the total concentration of competing agent. For the 1:1 direct competition of A and I for L in a mobile phase containing S, eq 5 predicts that a plot of 1/kA′ versus CI should yield a linear relationship, with the ratio of the slope to the intercept for this plot giving a value equal to KIL/(1 + KISCS). The values for some of the equilibrium constants in this system can be determined from eq 5 by preparing plots of 1/kA′ versus CI in the presence of several different levels of excess solubilizing agent. The ratio is then taken of the intercepts and slopes that are measured from these plots and a second graph is made of the intercept/slope ratio versus the concentration of solubilizing agent that was used in each individual study.

intercept/slope ) 1/KIL + (KISCS)/KIL Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

(6) 4603

According to eq 6, such a graph should give a linear relationship with an intercept equal to 1/KIL and a slope equal to KIS/KIL. By taking the reciprocal of the intercept obtained for this plot, the association equilibrium constant for the binding of I to L can be determined at the site of competition between A and I. In addition, by taking the ratio of new slope and intercept, the value of KIS can be obtained for the binding of I to the solubilizing agent. MATERIALS AND METHODS Reagents. The individual cis- and trans-clomiphene isomers were supplied by the Marion Merrell Dow Research Institute (Cincinnati, OH). β-Cyclodextrin, γ-cyclodextrin, racemic warfarin, L-tryptophan, cis/trans-tamoxifen, and HSA (Cohn fraction V, 99% pure, fatty acid free) were from Sigma (St. Louis, MO). The HSA and diol-bonded silica supports were prepared using Nucleosil Si-300 silica (7-µm particle diameter, 300-Å pore size) from Macherey-Nagel (Duren, Germany). Deuterium oxide (99.9% deuterated) and deuterated 2-propanol (99% deuterated) were from Cambridge Isotope Laboratories (Andover, MA). The 3-(trimethylsilyl)-1-propanesulfonic acid, sodium salt (>99% pure) was obtained from Aldrich (Milwaukee, WI). Other chemicals and biochemicals were of the purest grades available. All aqueous solutions in this study were prepared using water purified by a Nanopure water system (Barnstead, Dubuque, IA). Apparatus. An Omega 500 nuclear magnetic resonance spectrometer (GE NMR Instruments, Fremont, CA) was used to obtain the proton NMR spectra and to examine the stability of the clomiphene isomers. The chromatographic system consisted of a CM3000 solvent delivery system from LDC/Thermoseparations (Riviera Beach, FL), a Rheodyne 7010 injection valve (Cotati, CA) equipped with a pneumatic actuator from Valco (Houston, TX), a SM3100 UV/visible variable-wavelength detector from LDC, and a WOW data acquisition interface from LDC. Chromatograms were processed by programs written in Microsoft QuickBASIC (Redmond, WA). All columns and mobile phases were maintained at 37 ( 0.2 °C using an Isotemp 9100 water bath (Fisher Scientific, Pittsburgh, PA). The columns were downward slurry-packed using an HPLC column slurry packer from Alltech (Deerfield, IL). Stability and Solubilization of cis- and trans-Clomiphene. The sodium salt of 3-(trimethylsilyl)-1-propanesulfonic acid was used as the internal reference in the stability studies of cis- and trans-clomiphene by proton NMR. A 1 mM solution of the desired clomiphene isomer was made in a solvent mixture of 99.5% deuterium oxide and 0.5% deuterated 2-propanol. trans-Clomiphene dissolved easily in this solvent, but cis-clomiphene had to be sonicated for a long period of time before it dissolved. All samples used for this experiment were initially prepared in the dark and at 25 °C. Aliqouts of the samples were then taken and stored for up to 70 h in the absence or presence of normal room light and at various temperatures ranging from 4 to 37 °C. In the chromatographic studies, clomiphene and its isomers were dissolved by using β-cyclodextrin as a solubilizing agent in pH 7.4, 0.067 M phosphate buffer. The concentration of this solubilizing agent was varied from 1 to 2.6 mM during the zonal elution studies. Higher concentrations of β-cyclodextrin were not feasible because of its limited solubility in the phosphate buffer. β-Cyclodextrin concentrations lower than 1 mM were also not practical for use in this study since this prevented the effective 4604 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

dissolution of the cis- or trans-clomiphene that was present in the mobile phase. Chromatography. Diol-bonded silica was prepared as described previously.24 The diol coverage of the Nucleosil was 230 ( 3 µmol ((1 SD)/g of silica, as determined in duplicate by an iodometric capillary electrophoresis assay.25 The Schiff base immobilization method was used to couple HSA to the diol support.26 A bicinchoninic acid (BCA) protein assay was performed on the HSA support using HSA as the standard and diolbonded silica as the blank.27 The protein content was found to be 426 ( 2 nmol of HSA/g of silica. A 3 cm × 2 mm i.d. column was downward slurry-packed at 3500 psi with the immobilized HSA silica and enclosed in a water jacket for temperature control. A 1.0-2.6 mM concentration of β-cyclodextrin in pH 7.4, 0.067 M potassium phosphate buffer was used to prepare all mobile phases. To each of these solutions was added up to 10 µM of the desired competing agent (i.e., cis- or trans-clomiphene). After preparation, each mobile phase was filtered using a 0.22-µm nylon filter and degassed under vacuum for 15 min. The injected samples were then prepared using the same mobile phases employed in each respective study. The zonal elution experiments were performed at flow rates between 0.2 and 0.5 mL/min, with the desired probe being applied in replicate 20-µL injections. The column back pressure under these conditions typically ranged from 200 to 500 psi. Eluting samples of cis- or trans-clomiphene and cis/trans-tamoxifen were monitored at 268 nm; injected samples of (R)/(S)-warfarin were monitored at 306 nm. The retention time of each injected probe was calculated by using the first statistical moment of its corresponding peak.28 The void time of the column was determined by making similar injections with sodium nitrate, a nonretained compound. The capacity factor (k′) for the probe compounds was determined over a range of competing agent concentrations. Sample concentrations of 1-100 µM were tested for use in this study, with 100 µM being the typical level employed. There were no noticeable shifts in the capacity factors for these analytes over this range of sample concentrations, indicating that linear elution conditions were present, as assumed in eq 5. RESULTS AND DISCUSSION Stability and Solubilization of cis- and trans-Clomiphene. The long-term stability of clomiphene in aqueous solution was initially of concern since this compound is related to the same group of agents as stilbene, which is light sensitive. trans-Stilbene can undergo a trans/cis photoisomerization when irradiated with appropriate wavelengths of light; cis-stilbene can undergo a cis/ trans photoisomerization plus a photochemical reaction to form a phenanthrene-related compound.29 In this study, proton NMR was used to examine the stability of cis- and trans-clomiphene under various storage conditions in order to determine what conditions might be used to keep these isomers stable in their (24) Ruhn, P. F. Garver, S.; Hage, D. S. J. Chromatogr. 1994, 669, 9. (25) Chattopadhyay, A.; Hage, D. S. J. Chromatogr. 1997, 758, 255. (26) Larsson, P.-O. Methods Enzymol. 1984, 104, 212. (27) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Anal. Biochem. 1985, 150, 76. (28) Grushka, E.; Myers, M. N.; Schettlez, P. D.; Giddings, J. C. Anal. Chem. 1969, 41, 881. (29) Rodier, J.-M.; Myers, A. B. J. Am. Chem. Soc. 1993, 115, 10791.

Figure 2. Proton NMR spectra for (a) cis-clomiphene and (b) transclomiphene.

separate forms during the later chromatographic studies. It was found that the two isomeric forms could be differentiated by proton NMR in the region of 6.5-7.5 ppm (see Figure 2). NMR spectra were acquired for fresh aqueous solutions of cis- and transclomiphene before and after they had been stored for 70 h at 4-37 °C in either the presence or absence of normal room lighting. No noticeable conversion of cis- to trans-clomiphene or of transto cis-clomiphene was observed over this time frame for any of the storage conditions that were examined. This indicated that cis- and trans-clomiphene were sufficiently stable for use in chromatographic studies aimed at characterizing the HSA binding properties of the individual isomers. To avoid any chance of HSA denaturation due to the presence of an organic solvent such as 2-propanol, β-cyclodextrin was explored for use as an alternative agent to help solubilize cis- and trans-clomiphene in aqueous buffers. Such solubilization was required since the concentration of cis- and trans-clomiphene that could reliably be detected by a standard HPLC UV/visible absorbance detector or that was needed for a competing agent in the mobile phase was higher than the solubility limit of these compounds in the absence of any stabilizing agents. For example, when attempts were made to prepare a 10-4 M solution of clomiphene in pH 7.4 phosphate buffer, the result was an oily film that adhered to the walls of the glassware. However, 1-2.6 mM β-cyclodextrin was found to readily dissolve both cis- and transclomiphene at this concentration without any noticeable signs of insolubility. Absorbance and light-scattering measurements that were repeatedly performed on these solutions over the course of

Figure 3. Zonal elution plots of 1/k′ versus clomiphene concentration for (a) injections of cis-clomiphene in the presence of cisclomiphene as a competing agent and (b) injections of transclomiphene in the presence of trans-clomiphene as a competing agent. The solubilizing agent in both studies was 2.6 mM β-cyclodextrin. The dashed lines shown at the left are an extrapolation of the linear responses obtained at low mobile-phase concentrations of cis- and trans-clomiphene.

several days also failed to show any changes in the stability or solubility of the dissolved clomiphene. One advantage of using β-cyclodextrin as a solubilizing agent in this work is that it is hydrophilic agent that does not interact with HSA. This was confirmed by injecting a sample of β-cyclodextrin onto the HSA column in pH 7.4 phosphate buffer, which resulted in elution of β-cyclodextrin at the void time of the column. A similar absence of significant interactions between β-cyclodextrin and HSA (plus other serum proteins) was reported by Haginaka and Wakai for the injection of human serum onto a β-cyclodextrin column.30 The lack of interactions between β-cyclodextrin and HSA means that the presence of β-cyclodextrin in the mobile phase should not alter the equilibrium constants or nature of the binding that takes place between a small solute and HSA. Instead, the β-cyclodextrin and HSA would be expected to act as independent ligands, with the β-cyclodextrin binding to solutes through the formation of soluble host-guest complexes in the mobile phase (see reaction model given earlier in eqs 1-4). Competitive Binding Studies Using cis-Clomiphene as a Mobile-Phase Additive. The first set of zonal elution studies that was conducted in this work examined the competitive binding of injected cis- or trans-clomiphene in the presence of cisclomiphene as a mobile-phase additive. Figure 3a shows the typical response observed for plots of 1/kA′ versus CI for such (30) Haginaka, J.; Wakai, J. Anal. Chem. 1990, 62, 997.

Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

4605

Figure 4. Zonal elution results in the linear range of 1/k′ versus clomiphene concentration plots for the injection of (a) cis-clomiphene or (b) trans-clomiphene in the presence of cis-clomiphene as a competing agent and at several concentrations of β-cyclodextrin as a solubilizing agent. The concentrations of β-cyclodextrin were 1.00 (9), 1.50 (+), 2.00 (]), and 2.60 mM (4).

experiments over a wide range of clomiphene concentrations; Figure 4 gives a more detailed look at the results obtained at low clomiphene concentrations. In general, a linear region in these plots was observed for both cis- and trans-clomiphene when working at cis-clomiphene mobile-phase concentrations below 0.5 µM and at β-cyclodextrin concentrations of 1-2.6 mM. All further work was performed under these conditions. Regarding the nonlinear behavior seen at high clomiphene concentrations, one possible reason for this is that the total competing agent concentration was no longer negligible compared to the amount of solubilizing agent that was present in the mobile phase, as assumed in eqs 5 and 6. However, this should not have been a problem, since over a 10-fold molar excess of β-cyclodextrin versus clomiphene was used throughout this study. Another explanation is that the β-cyclodextrin was no longer able to solubilize all of the competing agent at the highest clomiphene concentrations that were used in Figure 3. As will be seen later, this is a likely reason for the observed nonlinearity, since both cis- and transclomiphene were found to have only weak binding to the β-cyclodextrin additive. As described in the Theory, the zonal elution-competitive binding studies were performed by using several concentrations of both cis-clomiphene and β-cyclodextrin as mobile-phase additives. When using 0-0.5 µM cis-clomiphene in the mobile phase, plots of 1/kA′ versus [cis-clomiphene] gave linear relationships with correlation coefficients ranging from 0.9997 to 0.9999 over four data points obtained at each β-cyclodextrin concentration (see Figure 4a). This linear behavior agrees with the results predicted 4606 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

Figure 5. Intercept/slope versus solubilizing agent concentration for the injection of (9) cis-clomiphene or (0) trans-clomiphene in the presence of (a) cis-clomiphene or (b) trans-clomiphene as a competing agent in the mobile phase.

by eq 5 for a 1:1 solute-ligand system when a large excess of solubilizing agent was used versus the competing agent in the mobile phase. A second graph was made by taking the ratio of the intercepts and slopes of the best-fit lines in Figure 4a and plotting these values versus the total, or analytical, concentration of β-cyclodextrin that was present in the mobile phase. The resulting graph is shown in Figure 5a. This plot also gave linear behavior, as predicted by eq 6, with a correlation coefficient of 0.9999 over the four concentrations of β-cyclodextrin that were used. From the reciprocal of the intercept in Figure 5a, it was possible to obtain the association equilibrium constant for the competition of cisclomiphene with itself at its binding regions on HSA. This gave a Ka value for this interaction of 7.4 ((0.2) × 106 M-1. The linearity of the graph in Figure 5a for competition between cisclomiphene and cis-clomiphene again supports a model in which cis-clomiphene is interacting at only one type of site on HSA. Similar competitive binding studies were performed by injecting small amounts of trans-clomiphene into the presence of cisclomiphene as a mobile-phase additive (see Figure 4b). Plots of 1/kA′ versus [cis-clomiphene] for these experiments gave linear relationships at competing agent concentrations below 0.5 µM, with correlation coefficients ranging from 0.9998 to 0.9999 over four data points obtained at each level of β-cyclodextrin. The ratio of the slopes and intercepts of these graphs were then plotted versus the total concentration of β-cyclodextrin in the mobile phase (see Figure 5a). The result was a linear relationship with a correlation coefficient of 0.9999 over four levels of β-cyclodextrin. The reciprocal of the intercept from this linear plot gave an association equilibrium constant 7.6 ((0.1) × 106 M-1 for the interaction of cis-clomiphene at the site at which it competes with

trans-clomiphene for HSA binding. This value was statistically identical to that obtained for cis-clomiphene/cis-clomiphene competition, indicating that the same site on HSA was involved in both competition processes. Furthermore, the linearity of the plots in Figures 4b and 5a indicate that cis- and trans-clomiphene had only one region on HSA at which they gave competitive binding. Competitive Binding Studies Using trans-Clomiphene as a Mobile-Phase Additive. The next series of studies examined the competition of injected trans-clomiphene with trans-clomiphene as an additive in the mobile phase. The overall behavior was similar to that seen for cis-clomiphene in Figures 4 and 5, in which linear behavior was observed in plots of 1/kA′ versus competing agent concentration at [trans-clomiphene] below 0.5-1.0 µM. The linear region of these curves for trans-clomiphene/trans-clomiphene competition gave correlation coefficients of 0.9995-0.9999 over the four data points generated at each level of β-cyclodextrin. The ratio of intercepts and slopes of these best-fit lines were then plotted as a function of β-cyclodextrin concentration, as shown in Figure 5b. The result, as seen earlier for cis-clomiphene, was a new linear relationship, as predicted by eq 6 for a system with 1:1 solute-ligand binding and in the presence of excess solubilizing agent. However, in this case, the data obtained from these plots now reflected the binding constants for trans-clomiphene, rather than cis-clomiphene, to HSA. From the reciprocal of the intercept in Figure 5b, an association equilibrium constant of 1.4 ((0.1) × 106 M-1 was obtained for the competition of transclomiphene with itself at its HSA binding regions. A fourth set of competitive binding studies was performed by injecting small amounts of cis-clomiphene into the presence of trans-clomiphene in the mobile phase. The plots of 1/kA′ versus [trans-clomiphene] for concentrations of 0-0.5 µM trans-clomiphene gave linear relationships with correlation coefficients of 0.9995-0.9999 over the four data points obtained at each concentration of β-cyclodextrin. Plots of the intercept/slope ratios of these best-fit lines versus the total concentration of β-cyclodextrin in the mobile phase gave a linear relationship with a correlation coefficient of 0.9976 over four β-cyclodextrin levels (Figure 5b). An association equilibrium constant of 1.2 ((0.1) × 106 M-1 was obtained from the inverse intercept of this plot for the interaction of trans-clomiphene at the cis-clomiphene binding site of HSA. Note that the Ka values obtained for trans-clomiphene in its competition with both cis- and trans-clomiphene were statistically identical. This indicates that the same binding region of HSA was involved in binding to both the cis and trans isomers of clomiphene. Competition of cis- and trans-Clomiphene with L-Tryptophan, Warfarin, and Tamoxifen. After it had been determined that both cis- and trans-clomiphene had a single binding site on HSA, zonal elution and HPAC were next used to help determine the location of this site by examining the competition of cis- and trans-clomiphene with other compounds that had known (or suspected) binding regions on HSA. For example, the interactions of cis- and trans-clomiphene at the major binding regions of HSA were studied by analyzing the competition of these compounds with L-tryptophan (a probe for the indole-benzodiazepine site of HSA) and (R)/(S)-warfarin (a probe for HSA’s warfarin-azapropazone site).8-10 Experiments performed with L-tryptophan as a competing agent did not show any significant

Figure 6. Zonal elution results for the injection of (a) racemic warfarin and (b) cis/trans-tamoxifen in the presence of cis-clomiphene (]) or trans-clomiphene (9) as a competing agent in the mobile phase. The concentration of β-cyclodextrin in the mobile phase was 2.6 mM. The tamoxifen sample contained 100 µM cis/trans-tamoxifen and 3.8 mM γ-cyclodextrin in 0.067 M, pH 7.4 phosphate buffer; the warfarin sample contained 65 µM (R)/(S)-warfarin in pH 7.4 phosphate buffer. Other conditions were the same as in the clomiphene/ clomiphene competitive binding studies.

changes in the retention times for either cis- or trans-clomiphene (i.e., only random variations in kA′ of 7% or less for mobile phases containing 0-50 µM L-tryptophan). This indicated that the binding region for cis- and trans-clomiphene was not located at the indole-benzodiazepine site on HSA. The interactions of cis- and trans-clomiphene at the warfarinazapropazone site were initially studied by injecting small amounts of these compounds into the presence of 0-10 µM (R)/(S)warfarin in the mobile phase. Under these conditions, the capacity factors for both cis- and trans-clomiphene showed a significant increase as the mobile phase concentration of warfarin was raised. The reverse experiment, in which warfarin was injected into the presence of cis- or trans-clomiphene in the mobile phase, produced similar results in which the capacity factor for warfarin increased (or 1/kA′ decreased) with an increase in clomiphene concentration (see Figure 6a). This behavior indicates that cis- and transclomiphene have indirect, or allosteric, competition with warfarin and that these agents do not bind directly at the warfarinazapropazone site of HSA. Similar positive allosteric effects have been reported for HSA in solution-phase studies examining the competition between warfarin and tamoxifen, a compound that is closely related to cis/trans-clomiphene in structure.23 The last series of studies looked at the competition of cis- and trans-clomiphene with tamoxifen, which has been suggested to have a binding region on HSA that is separate from the warfarinAnalytical Chemistry, Vol. 70, No. 21, November 1, 1998

4607

azapropazone or indole-benzodiazepine sites.23 It has been found in previous solution-phase studies that cis/trans-clomiphene and cis/trans-tamoxifen do share common regions for their interactions on HSA,23 but the actual number of these common binding sites has not been thoroughly studied. This was examined in this work by using the same zonal elution methods as described earlier for examining the competition of cis- and trans-clomiphene with each other. The only modification that was required was the use of γ-cyclodextrin instead of β-cyclodextrin as a solubilizing agent for placing tamoxifen into solution. Figure 6b shows some typical results from these zonal elution studies. Linear plots were obtained for plots of 1/kA′ versus [clomiphene] for the injection of cis/trans-tamoxifen into the presence of either cis- or transclomiphene. The correlation coefficients of these plots were 0.9994-0.9997 over four clomiphene concentrations in the range of 0-1.0 µM. This result confirms that tamoxifen has 1:1 competition with cis- and trans-clomiphene; this also indicates that the clomiphene-binding region that was analyzed throughout this work is the same as the tamoxifen site of HSA that has been proposed in earlier studies.23 Binding of cis- and trans-Clomiphene to β-Cyclodextrin. According to eq 6, the plots shown in Figure 5 should not only provide the value of Ka for the binding of the competing agent to the immobilized ligand but should also provide the equilibrium constant (KIS) that is involved in the binding of the competing agent to the solubilizing agent (i.e., β-cyclodextrin). This can be obtained from the slope/intercept ratio of such graphs. When this was done for the cis-clomiphene/cis-clomiphene data, the resulting KIS value for the binding of cis-clomiphene to β-cyclodextrin was found to be 3.0 ((0.5) M-1. From the transclomiphene/cis-clomiphene studies, a similar value of 4.6 ((0.5) M-1 was obtained for the interactions of cis-clomiphene with β-cyclodextrin. The fact that linear relationships were observed in Figure 5a for both determinations supports a model in which cis-clomiphene and β-cyclodextrin formed a 1:1 complex under the conditions used in this study. Solubilization constants were also determined for trans-clomiphene from the zonal elution studies in which this compound was used as the competing agent. For example, from the transclomiphene/trans-clomiphene data in Figure 5b, a value of 0.3 ((0.1) M-1 was calculated for the interactions of trans-clomiphene with β-cyclodextrin. Similarly, from the cis-clomiphene/transclomiphene studies, a value for KIS of 0.6 ((0.1) M-1 was obtained for trans-clomiphene and β-cyclodextrin. The linear behavior noted in Figure 5b indicates that trans-clomiphene and β-cyclodextrin were also forming a 1:1 complex under the experimental conditions that were used in this work. Although these KIS values are quite small, binding constants of a similar size have been reported for the interactions of several other agents with cyclodextrins.31 These binding interactions, though weak in nature, were still apparently sufficient to bring the solubility of the cis- and trans-clomiphene up to acceptable levels. However, this weak binding does explain why β-cyclodextrin concentrations down to only ∼1.0 mM could be used to successfully bring enough clomiphene into solution to perform the chromatographic studies. In addition, these weak interactions (31) Palepu, R.; Richardson, J. E.; Reinsborough, V. C. Langmuir 1989, 5, 218.

4608 Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

Table 1. Association Equilibrium Constants for the Competitive Binding of cis- and trans-Clomiphene at Their Binding Sites on HSA at pH 7.4 and 37 °C competing agent and association constant (×10-6 M-1) for competing agent at its site of competition with the injected probea

cis-clomiphene trans-clomiphene av assoc const for competing agent

cis-clomiphene

trans-clomiphene

7.4 ((0.2) 7.6 ((0.1) 7.5 ((0.2)

1.2 ((0.1) 1.4 ((0.1) 1.3 ((0.2)

a The numbers in parentheses represent a range of (1 SD. All of the above values were determined in 0.067 M potassium phosphate buffer.

may explain why nonlinear behavior was seen in Figure 3 and related plots when using clomiphene concentrations above 0.5 µM. SUMMARY This study examined the use of HPAC and zonal elution to examine the interactions between proteins and solutes that are relatively insoluble in aqueous media. Part of this work involved the extension of current zonal elution theory to include the case in which a solubilizing agent is present in the mobile phase to aid in the dissolution of the competing agent or injected analyte. Equations based on this theory were then used for the determination of binding constants between cis- and trans-clomiphene and immobilized HSA in the presence of solution-phase β-cyclodextrin. The same equations and theory should be useful when HPAC is employed for the study of any other compounds with limited solubility or for the more general situation in which independent immobilized and soluble ligands are competing for solute binding. On the basis of the tools that were developed in this report, it was found that cis- and trans-clomiphene each have 1:1 interactions at a common binding region on HSA. Further experiments indicated that this binding region was the same as the tamoxifen site that has been proposed in earlier solution-phase studies.23 The association equilibrium constants that were measured for cis- and trans-clomiphene at this site are summarized in Table 1. Both compounds had association constants that were in the range of 106-107 M-1, with cis-clomiphene having 5-6 times stronger binding to HSA than trans-clomiphene at physiological pH. Although no association constants have previously been reported for HSA with the separate isomers of clomiphene, the size of these values is comparable to those that have been reported for the 1:1 interactions of several other compounds that bind to HSA, such as (R)/(S)-warfarin and (R)/(S)-ibuprofen.8,16 The results provided in this study demonstrate the types of information that can be obtained by HPAC and indicate the potential value of this method in characterizing the interactions between proteins and drugs or other solutes. ACKNOWLEDGMENT This work was supported under Grant RO1 GM44931 from the National Institutes of Health. APPENDIX Derivation of Eq 5. The following equilibrium expressions can be written for the reactions given earlier in eqs 1-4,

KAL ) [AL]/([A][L])

(A1)

KIL ) [IL]/([I][L])

(A2)

phase at equilibrium (i.e., at the true center of the chromatographic peak for A). This can be written in the form of eqs A13 and A14 for the reaction scheme shown in eqs 1-4. By

KAS ) [AS]/([A][S])

(A3)

kA′ ) mAL/(mA + mAS)

(A13)

KIS ) [IS]/([I][S])

(A4)

kA′ ) (mAL/mA)/(1 + mAS/mA)

(A14)

where [ ] represents the actual or apparent molar concentration of each species in the column. Rearrangement of eqs A1-A4 gives rise to the following alternative relationships.

substituting eqs A5, A7, and A11 into the above relationship, the following equation is obtained.

kA′ ) (KAL[L])/(1 + KASCS)

(A15)

KAL[L] ) [AL]/[A] ) mAL/mA

(A5)

KIL[L] ) [IL]/[I] ) mIL/mI

(A6)

Combining eqs A12 and A6 gives the expression shown for [L] in eq A16. In a similar fashion, the value of [I] can be related to

KAS[S] ) [AS]/[A] ) mAS/mA

(A7)

[L] ) CL/(1 + KIL[I])

KIS[S] ) [IS]/[I] ) mIS/mI

(A8)

where mA, mAL, mIL, mI, mAS, and mIS represent the moles of each of the indicated species that are present at equilibrium. The total concentrations of the solubilizing agent and ligand that are present in the column can be described by the mass balance expressions shown in eqs A9 and A10. If it is assumed

CS ) [S] + [AS] + [IS]

(A9)

CL ) [L] + [AL] + [IL]

(A10)

(A16)

the total concentration of I that is present in the mobile phase (CI) through eq A8 and the relationships shown in eqs A17 and A18. Substitution of eq A18 into eq A16 provides the following

CI ) [I] + [IS]

(A17)

[I] ) CI/(1 + KISCS)

(A18)

[L] ) CL(1 + KISCS)/(1 + KISCS + KILCI)

(A19)

equation for [L].

that the amount of A that is injected is much smaller than the number of ligand sites in the column (i.e., linear elution conditions are present) and that S is present in a large excess versus A and I, then eqs A9 and A10 are approximately equal to the following expressions.

Substituting eq A19 into eq A15 results in the following expression.

CS ) [S]

(A11)

Taking the reciprocal of this last equation then provides the final relationship given in eq 5.

CL ) [L] + [IL]

(A12)

By definition, the capacity factor kA′ will be equal to the moles of A that are present in the stationary phase versus the mobile

kA′ ) KALCL(1 + KISCS)/[(1 + KASCS)(1 + KISCS + KILCI)] (A20)

Received for review July 7, 1998. Accepted August 25, 1998. AC980734I

Analytical Chemistry, Vol. 70, No. 21, November 1, 1998

4609