Anal. Chem. 1998, 70, 373-377
Quantitative Study of Competitive Binding of Drugs to Protein by Microdialysis/High-Performance Liquid Chromatography Hailin Wang, Hanfa Zou,* and Yukui Zhang
National Chromatographic R.&A. Centre, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China
A displacement equation describing competitive binding of drugs to protein in solution is derived and examined with four nonsteroidal anti-inflammatory drugs and human serum albumin as model drugs and protein, respectively. Microdialysis/high-performance liquid chromatography was adopted to determine simultaneously the unbound solute and displacing agent in drug-protein solutions. The method is able to locate the binding site and determine affinity constants even up to 107 L/mol accurately. A comparison of association constants determined by this method and from capacity factors on HSACSP is given. Most drugs undergo some degree of reversible binding to plasma proteins, a process which may have significant effects on the overall activity profile of the compounds.1,2 Various methods are used to evaluate protein and drug interactions,3-5 but they suffer from a number of problems in their ability to examine the drugs highly bound to protein. For conventional biochemical techniques (i.e., ultrafiltration, equilibrium dialysis), the amount of unbound drug is very low and difficult to detect, except with enrichment by solid phase extraction or use of radiolabeled drug; it is also subject to significant error(s) when impurities are present in the drug used in the protein-binding experiments. Protein (i.e., serum albumin and R1-acid glycoprotein) chiral stationary phases (CSPs) have been employed to probe the interaction of protein and drug,6,7 but they require that the mobile phase include a small percentage of organic solvents, for instance, 2-propanol and acetonitrile, which may not accurately reflect drug-protein equilibrium in aqueous solution, to allow the solute to elute out from the column within an accepted time. It is necessary to develop a sensitive and simple method to determine the interactions between high-affinity drugs and proteins to advance this kind of study. Displacement studies with endogenous and/or exogenous compounds as competitive agents have been utilized to provide (1) Kwing, T. C. Clin. Chim. Acta 1985, 148, 151-216. (2) Svensson, C. K.; Woodruff, M. N.; Baxter, J. G.; Lalka, D. Clin. Pharmacokinet. 1986, 11, 450-469. (3) Sebile, B. Fundam. Clin. Pharmacol. 1990, 4, (Suppl. 2), 151s. (4) Scholtan, W. Arzeneim.-Forsch. 1965, 15, 1433. (5) Shibukawa, A.; Nishimura, N.; Nomura, K.; Kuroda, Y.; Nakagawa, T. Chem. Pharm. Bull. 1990, 38, 443. (6) Kaliszan, R.; Nasal, A.; Turowski, M. Biomed. Chromatogr. 1995, 9, 211. (7) Nilsson, K.; Larsson, P.-O. Anal. Biochem. 1983, 134, 60. S0003-2700(97)00564-7 CCC: $15.00 Published on Web 01/15/1998
© 1998 American Chemical Society
additional information on the mechanism of the binding and possible interactions and to help in the further characterization of drug and macromolecule interactions. Such information is of special importance, especially to improve the safety of therapy with combinations of drugs that are highly protein bound and might compete for binding sites. It is well known that competitive binding of cobinding solutes to a protein will elevate free concentrations of both drugs, which may be easy to detect accurately. In this work, a displacement equation is derived and examined by microdialysis/HPLC, which has been applied to determine the molecular interactions of various drugs and human serum albumin (HSA).8-11 This method has a number of advantages: it is fast, simple, and easily automated and hyphenated with other analytical techniques. Now, the method is used to validate the quantitative relationship between free drugs in competitive binding of drugs to protein. Fenoprofen, ketoprofen, and naproxen were chosen as solutes and ibuprofen was chosen as displacing agent because they are synthetic nonsteroidal anti-inflammatory drugs (NSAIDs) that are often coadminstered with other drugs and are highly bound to HSA. Human serum albumin was chosen because it is a principal binding protein in plasma for a number of drugs as well as some endogenous compounds (i.e., bilirubin, fatty acids, etc.). THEORETICAL SECTION The derivation of the displacement equation is based on two assumptions, which have been supported by a large number of observations, as follows: (1) Drugs bind to a protein according to the site-binding model. Two drugs binding to the same sites of a protein will elevate the free fractions of both drugs. (2) The binding of drugs to a protein conforms to the stoichiometric relationship. A drug, A, could be bound to two type-independent binding sites of a protein, P: one is the primary binding site, with fewer sites on one protein molecule and a higher affinity for drug A; the other is the secondary binding site, with more sites on one (8) Wang, H. L.; Zou, H. F.; Zhang, Y. K. Chromatographia 1997, 44, 205208. (9) Wang, H. L.; Zou, H. F.; Feng, A. S.; Zhang, Y. K. Anal. Chim. Acta 1997, 342, 159-165. (10) Wang, H. L.; Zou, H. F.; Zhang, Y. K. Biomed. Chromatogr. 1997, 11, 4. (11) Wang, H. L.; Zou, H. F.; Zhang, Y. K. Sci. China Ser. B 1997, 27, 6.
Analytical Chemistry, Vol. 70, No. 2, January 15, 1998 373
protein molecule and a lower affinity for drug A. For drug A and protein P in aqueous solution, there are two independent binding reactions: KA
A + P1 98 AP1 K2
A + P2 98 AP2
(1)
K2 )
[AP]1 [A][P]1 [AP]2 [A][P]2
(3)
(4)
In eqs 3 and 4, K1 and K2 are the association constants for the binding of A to the primary and secondary binding sites of the protein P, respectively. The term [A] represents the free concentration of A, [P]1 and [P]2 represent the free concentrations of primary and secondary binding sites on the protein P, while [AP]1 and [AP]2 represent the bound concentrations of A to the sites of P1 and P2, respectively. If a displacing agent, B, which shows a high affinity for the primary binding sites of the protein, P1, is added to the mixed solution of A and the protein P, then an additional binding reaction occurs: KB
B + P1 98 BP1
(5)
[AP]1 ) K1[A][P]1
(6)
[BP]1 ) KB[B][P]1
(7)
The total concentration of the primary binding sites of the protein P, CP,1, is constant according to the mass action law; therefore, an expression for CP,1 is obtained:
(8)
By substituting [AP]1 and [BP]1 from eqs 6 and 7 into eq 8, the following relationship is obtained:
K1[A][P]1 + KB[B][P]1 + [P]1 ) CP,1
(9)
Then,
[P]1 )
CP,1 1 + K1 [A] + KB [B]
(10)
By substituting eq 10 into eq 6, it can be converted to eq 11: 374
Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
K1CP,1 1 + K1[A] + KB[B]
(11)
By taking the reciprocal of both sides of eq 11, the following equations are obtained:
KB [A] [A] 1 ) + + [B] [AP]1 K1CP,1 CP,1 K1CP,1
(12)
KB [A] [A] 1 ) + [B] [AP]1 CP,1 K1CP,1 KACP,1
(13)
According to assumption 2, the binding of drugs to protein conforms to the stoichiometric relationship, so the numbers of primary and secondary binding sites on one protein molecule are positive integers; therefore, eqs 14 and 15 are obtained:
CP,1 ) n1CP
(14)
CP,2 ) n2CP
(15)
where CP represents the total concentration of protein P, n1 and n2 are the numbers of primary and secondary binding sites on one protein P molecule, respectively, and CP,2 represents the total concentration of secondary binding sites. The secondary bound concentration of the solute A, [AP]2, independent of primary binding and unaffected by the displacing agent B, is influenced only by the unbound concentration of the solute A. From eq 4, an expression for [AP]2 is obtained:12
[AP]2 )
From eqs 1 and 5, eqs 6 and 7 can be obtained as follows:
[AP]1 + [BP]1 + [P]1 ) CP,1
[A]
) K1[P]1 )
(2)
where P1 and P2 represent the primary and secondary binding sites of protein P, so AP1 and AP2 represent the complexes of A with the protein at the sites of P1 and P2, respectively. Their equilibrium equations can be expressed as follows:
K1 )
[AP]1
n2K2CP[A] 1 + K2[A]
(16)
where K2 represents the secondary association constant of drug A to protein P. By the mass action law, an expression for the bound concentration of A is obtained:
[AP]1 + [AP]2 ) [AP]
(17)
where [AP] represent the total bound concentration of the solute A. Equation 17 can be converted to eq 18 as follows:
[A] [A] 1 (18) ) ) [AP]1 [AP] - [AP]2 [AP]/[A] - [AP]2/[A]
The following expression can be derived by relating [AP]1/[A] to [B]. [AP]/[A] is defined as k′ and named the apparent capacity factor. By substituting eqs 14, 16, and 18 into eq 13, therefore, an equation describing the complex competitive binding of two drugs to protein is obtained: (12) Feldman, H. A. Anal. Biochem. 1972, 48, 317.
k′ -
KB [A] 1 1 ) + [B] (19) n2K2CP n1CP K1n1CP K1n1CP 1 + K2[A]
A new parameter, X0, to simplify the displacement equation, is defined as
X0 )
n2K2CP
(20)
1 + K2[A]
By substituting eq 20 into eq 19, it is converted to eq 21 as follows:
KB [A] 1 1 ) + [B] k′ - X0 n1CP K1n1CP K1n1CP
(21)
In most cases, one protein molecule has just one primary binding site, which means that n1 ) 1, then eq 21 is simplified as
KB [A] 1 1 ) + [B] k′ - X0 CP K1CP K1CP
(22)
Equation 22 describes a complex and general case in the binding of drug to protein, in which the two drugs competitively bind to the primary binding site on a protein, but only the solute binds to the secondary binding sites, unaffected by the displacing agent. There are other cases in the binding of drug to protein. One is the simplest is that in which the two drugs competitively bind to a single site without solute binding at the secondary site, which means n2 ) 0. Then, eq 22 is simplified as
KB 1 [A] 1 ) + [B] k′ CP K2CP K2CP
(23)
On the other hand, in addition to the binding of drug A to the secondary sites of a protein, there are other minor binding events, which are also unaffected by competitor. Then X0 will extend to m
X0 )
∑n K C /(1 + K [A]) + n i
i P
i
m+1Km+1CP
(24)
i)2
where m is the number of classes of independent adsorption sites, ni is the number of sites in a class i with an association constant of Ki, and nm+1Km+1CP represents nonspecific adsorption of A on the protein surface. Finally, if there are three or more drugs competitively binding to the primary binding site on protein, an equation can also be obtained based on an analogous treatment for eq 22, as follows:
KB KC [A] 1 1 ) + [B] + [C] + ‚‚‚ k′ - X0 CP KACP KACP KACP
(25)
In eq 25, C, ‚‚‚ represents the additional competing agents, and KC, ‚‚‚ represents the association constants of C, ‚‚‚. This equation is useful for dealing with the competitive binding of multiple drugs
to a protein and in evaluating the association constants of multiple drugs simultaneously by nonlinear regression. In general, the association constants of minor sites is very low, about 104 L/mol or even lower. In the competitive binding study, the total concentration of A is constant and [A] is very low; then 1 + Ki[A] ≈ 1.0, and X0 might be simplified as a constant. In drug and protein mixed solutions, if there are two drugs competitively bound to the protein, one of the eqs 22 and 23 might be applied, and then a plot of 1/(k′ - X0) - [A]/CP against [B] will be linear. The slope divided by the intercept will give KB, which is the affinity constant for the binding of the competing agent to the site in question; the intercept is equal to 1/KACP, so the affinity constant of solute A can also be obtained from the intercept. If three or more drugs are cobinding to a protein in solution, eq 25 might be applied to obtain the interaction parameters of the drugs to the protein. EXPERIMENTAL SECTION Reagent and Materials. Fenoprofen, ketoprofen, naproxen, ibuprofen, and HSA (fatty acid and globulin-free) were purchased from Sigma (St. Louis, MO). A CMA/20 microdialysis probe was purchased from CMA/Microdialysis (Acton, MA); the length of the dialysis membrane is 4 mm. Preparation of the Sample Solution. The drug stock solutions were made up in ethanol with concentrations of 10 mmol/L. HSA was dissolved in a potassium phosphate buffer (pH 7.4, 0.067 mol/L) at a concentration of 50 µmol/L. Appropriate volumes of one drug and ibuprofen stock solutions were put in a 2 mL open vial by using an electric pipet, and the ethanol was evaporated in air. Next, 1.5 mL of the prepared HSA solution (in phosphate buffer of pH 7.4, 0.067 mol/L) was added to the vial to obtain the mixture solutions of drugs with HSA. Microdialysis Sampling. The microdialysis system comprises a FAMILIC-100N microsyringe pump (JASCO, Kyoto, Japan) and a microdialysis probe (CMA). The perfusion solution is a 0.067 mol/L potassium phosphate buffer of pH 7.4; the perfusion rate is 1 µL/min. The microsyringe was filled with the perfusion solution. The drugs-HSA mixed solution was incubated at 37 °C in a water bath for more than 10 min before the probe was put into this solution, and sampling from the solutions was started. After 12 min, the microdialysate was collected for 30 min. The collected microdialysate was handled for LC analysis. The probe must be washed by the perfusion solution at a rate of 5 µL/min for several minutes before the probe is put into the mixed solution to get rid of air in the probe and of organic solvents, which are used for protection of the dialysis membrane. Apparatus and Instruments. The present LC system comprises a LC-10A pump (Shimadzu, Kyoto, Japan), a Rheodynetype injector valve with a 20 µL loop, a SPD-10AV UV detector (Shimadzu), and a WDL-95 chromatographic workstation (National Chromatographic R.&A. Centre, Dalian, China). The column used is 4.0 mm i.d. × 100 mm, packed with 5 µm particles of Spherisorb C18 (packed by the National Chromatographic R.&A. Centre). The mobile phase used was methanol/water/ acetic acid (72.5:27.5:1.0), and the solutes were detected at UV 232 nm. The flow rate of the mobile phase was 1.0 mL/min. Recovery of Microdialysis. The recovery of microdialysis sampling (R), also called the microdialysate extraction fraction, Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
375
Table 1. Recovery of Four NSAIDs by Microdialysis Sampling ketoprofen recovery (%)
43.37
fenoprofen 56.03
naproxen 51.56
Table 2. Parameters Obtained from the Displacement of NSAIDs by Ibuprofena
ibuprofen 45.22
solute
X0
intercept
slope
γ
K1 (L/mol)
KB (L/mol)
ketoprofen 1.162 0.017 46 0.009 321 0.9983 1.21 × 106 5.34 × 105 fenoprofen 2.2182 0.008 216 0.007 299 0.9982 2.43 × 106 8.88 × 105 naproxen 1.693 0.002 841 0.002 495 0.9976 7.00 × 106 8.78 × 105 a X is the contribution of k′ to binding at sites unaffected by 0 ibuprofen; columns 2 and 3 summarize the slope and intercept of the regression curves, respectively; γ is the correlation coefficient; K1 is the affinity constant of the solutes for the site from which ibuprofen displaces it; KB is the affinity constant of ibuprofen for the site from which it displaces the NSAIDs.
Figure 1. Chromatogram of microdialysate sampled from the 30 µmol/L fenoprofen and 50 µmol/L human serum albumin mixed solution with addition of 25 µmol/L ibuprofen as displacing agent. Fenoprofen and ibuprofen in the microdialysate are of 0.59 ( 0.02 and 1.24 ( 0.04 µmol/L, respectively. Detection wavelength is 232 nm; for other experimental conditions, see the text.
is defined as the concentration ratio of the drug in microdialysate (Cd) to the unbound fraction in drug-protein solution. The microdialysate was collected by microdialysis sampling in a standard solution with 0.067 mol/L potassium phosphate buffer of pH 7.4 and then analyzed by RP-LC. The standard solution of drugs was also analyzed by RP-LC. The recovery of individual drug was calculated from the peak area ratio of drug in microdialysate to that in standard drug solution. RESULTS AND DISCUSSION Each of ketoprofen, fenoprofen, and naproxen has been used as solute with ibuprofen used as displacing agent, and their free concentrations were determined by means of microdialysis/HPLC. The free drug concentrations (Cf) in drug-HSA mixed solution can be calculated as follows:
Cf ) Cd/R
The recovery (R) is a key parameter in the microdialysis method for the determination of drug-protein interaction. Our previous works demonstrate recovery of microdialysis sampling to determine the free drug reproducibly and precisely.8,9,13 In this study, the recoveries of four nonsteroidal anti-inflammatory drugs (NSAIDs) are obtained and listed in Table 1. A typical chromatogram of microdialysate sampled from fenoprofen and HSA mixed solution including ibuprofen with the lowest free concentrations of drug and displacing agent is presented in Figure 1. The HPLCcombined ultraviolet detector makes it easy to determine the drugs in the microdialysates, even at the lowest concentration on the experimental conditions because of the displacing effect. (13) Wang, H. L.; Zou, H. F.; Feng, A. S.; Zhang, Y. K. Chinese J. Anal. Chem. 1997, 25, 212.
376 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
Figure 2. Influence of ibuprofen on the binding of ketoprofen (4), fenoprofen (0) and naproxen ([), plotted according to a model describing competition at a single site with further binding of the solute at other site(s), which are unaffected by ibuprofen. X0 is the contribution to k′ from such unaffected sites.
In the competitive binding experiment, the total concentrations of drug used as solute and HSA are constant. Increasing concentrations of ibuprofen, used as competing agent, were added to the test solute and HSA solution, and the displacement of all test solutes (measured as an increase in free concentration) was followed. Table 2 shows the interaction parameters obtained from the displacement of the NSAIDs by ibuprofen after having plotted 1/(k′ - X0) - [A]/CP versus [B] (free concentration of ibuprofen in this study) by employing eq 22. All compounds tested gave linear relationships, as shown in Figure 2. Linear regression analysis on the data gave correlation coefficients (γ) of 0.9976 and above for all compounds. X0 can be obtained by an iterative test, which reveals the contribution to k′ from the sites unaffected by ibuprofen. The affinity constant of ibuprofen (KB) for the sites from which it displaced the NSAIDs was obtained by calculating the ratio of the slope to the intercept for each curve; KB is about (7.7 ( 1.6) × 105 L/mol. The primary association constants of the solutes were obtained from the intercept because CP is known, and they are also listed in Table 2. The total affinity constants of secondary binding were calculated from X0 and are listed in Table 3. Ketoprofen, fenoprofen, and naproxen were significantly displaced by ibuprofen, confirming that they bind to indolebenzodiazepine sites of HSA (site II). In addition, data from Table 2 reveal that the values of X0 are not equal to zero, suggesting that they bind to the secondary sites on HSA in addition to the
Table 3. Secondary Affinity Constants of Solutes Calculated from X0 Obtained from the Displacement Equation
n2K
(×104
L/mol)
ketoprofen
fenoprofen
naproxen
2.4
4.4
3.8
Figure 3. Plot of the drug-HSA association constant, K, determined by plotting the displacement equation against the capacity factor, k′, for HSA-CSP, obtained from ref 14. ([) Data on line (K ) (-7.1 × 104) + (1.26 × 105) L/mol, γ ) 0.9986); (2) data deflect seriously from the line.
primary binding site. The association constants of ketoprofen, fenoprofen, and naproxen correlated very well with their capacity factors on HSA-CSP, as shown in Figure 3, which were proportional to the total affinity of the drugs, suggesting that the sites from which ibuprofen displaces them are their primary binding sites on HSA. Chromatographic retention data are obtained from ref 14. However, ibuprofen deflects the linear relationship seriously, suggesting that the displacements occur at lower affinity sites on HSA. Our results agree well with those of Rahim and Aubry,15 who reported that site II of HSA may be composed of two subsites to which (R)-ibuprofen binds with higher and lower affinity, and (R)-ibuprofen displaces fenoprofen A from the lowaffinity site. It can be concluded that ketoprofen, fenoprofen, naproxen, and ibuprofen predominantly bind to indole and benzodiazepine sites of HSA (site II). Maybe the sites from which ibuprofen displaces ketoprofen, fenoprofen and naproxen are lower affinity subsites of site II. Equation 23, which describes simple, single-site competition, was unsuccessful in fitting the actual experimental data, obtained for the present solutes and ibuprofen, indicating that the processes (14) Noctor, T. A. G.; Felix, G.; Wainer, I. W. Chromatographia 1991, 31, 55. (15) Rahim, S.; Aubry, A.-F. J. Pharm. Sci. 1995, 84, 949. (16) Joshi, A. S.; Pieniaszek, H. J., Jr.; Quon, C. Y.; King, S.-Y. P. J. Pharm. Sci. 1994, 83, 1187.
involved were complex. However, eq 22, which includes a term for solute binding at the sites which are unaffected by the competitor, was able to very well describe the behavior of four NSAIDs competitively binding to HSA. In this study, microdialysis/HPLC was used to determine the unbound solute and competing agent simultaneously. Other conventional methods, such as ultrafiltration and equilibrium dialysis combined with HPLC, also have the ability to study the competitively binding experiments quantitatively and evaluate the interactions of drugs and proteins with high affinity precisely by employing the displacement equation. Equation 25 might be applied to study the competitive binding of three or more drugs to protein and to evaluate the interactions of three or more drugs with protein simultaneously. In future studies, we will examine this possibility by a multidrug competitive binding experiment. The primary association constants of ketoprofen and fenoprofen are similar, which are determined directly by the conventional method, and that of naproxen is slightly higher than those of fenoprofen and ketoprofen, but their capacity factors on HSACSP are 2 times higher. This phenomenon is contradictory. The direct determination of the interaction of drug and protein with high affinity by conventional methods may be subject to significant errors because the unbound drug concentrations is very low and trace impurities interfere with the measurement.16 The method described shows a number of advantages as follows: HPLC analysis is an easy and convenient way to detect the unbound concentrations of both solute and displacing agent; this method is able to simultaneously and quickly estimate the equilibrium constants of the two drugs; able to locate binding sites on HSA; and it is suitable to apply it to microdialysis/HPLC as well as ultrafiltration and equilibrium dialysis, which have been adopted widely. CONCLUSION The displacement equation, which describes competition of two drugs at a single site, with solute binding at secondary site(s), unaffected by competing agent, can be applied to determine the interaction of high-affinity drug and protein with a number of advantages: it is simple, sensitive and precise. Ketoprofen, fenoprofen, naproxen, and ibuprofen predominantly bind to the site II on HSA with very high affinity; maybe the sites from which ibuprofen displace NSAIDs are lower affinity subsites of site II. ACKNOWLEDGMENT Financial support from the administration of the Chinese Academy of Sciences and the National Science Foundation of China to Dr. Hanfa Zou is gratefully acknowledged. Received for review June 2, 1997. Accepted October 7, 1997.X AC970564U X
Abstract published in Advance ACS Abstracts, November 15, 1997.
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