INTERACTION BETWEEN DRUGS AND BIOMEDICAL MATERIALS. I. BINDING POSITION OF BEZAFIBRATE TO HUMAN SERUM ALUBMIN

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1 Advanced Materials Development and Performance (AMDP2011) International Journal of Modern Physics: Conference Series Vol. 6 (2012) World Scientific Publishing Company DOI: /S INTERACTION BETWEEN DRUGS AND BIOMEDICAL MATERIALS. I. BINDING POSITION OF BEZAFIBRATE TO HUMAN SERUM ALUBMIN MASAMI TANAKA Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima , Japan, tanaka@ph.bunri-u.ac.jp KEIJI MINAGAWA Institute of Technology and Science, The University of Tokushima, Tokushima , Japan, minagawa@chem.tokushima-u.ac.jp MOHAMED R. BERBER Department of Chemistry, Faculty of Science, Tanta University, Tanta 31527, Egypt, INAS H. HAFEZ Institute of Technology and Science, The University of Tokushima, Tokushima , Japan, TAKESHI MORI Faculty of Engineering, Kyushu University, Fukuoka, , Japan, mori.takeshi.880@m.kyushu-u.ac.jp The interaction between bezafibrate (BZF) and human serum albumin (HSA) was investigated by equilibrium dialysis. Since the binding constant of BZF to HSA was independent of ionic strength and decreased with the addition of fatty acid, the interaction between BZF and HSA was considered to be due to hydrophobic mechanism. Chemical shifts in 1 H-NMR spectra of BZF were independent of the concentration of BZF and addition of HSA. Spin-lattice relaxation time (T 1 ) and spin-spin relaxation time (T 2 ) of respective protons of BZF were independent of the concentration, but depended on the concentration of HSA added. The binding position of BZF to HSA was considered to involve the hydrophobic aromatic moiety of BZF from the ratio of spinspin relaxation rates (1/T 2 ) of BZF bound to HSA and free BZF. Keywords: Hydrophobic interaction; bezafibrate; human serum albumin; spin-spin relaxation rate. It is important to study the antagonism of drugs binding to albumin in order to clarify their side effects accompanying the increase of drug concentration. An antihyperlipidemic drug Bezafibrate (BZF; 2-(4-{2-[(4-Chlorobenzoyl)amino]ethyl} phenoxy)-2-methylpropanoic acid) is known to interact with other drugs at combined use. Its binding to albumin has been mainly discussed based on the binding ratio and other macroscopic changes 1). However, the microscopic behaviors such as the binding 751

2 752 M. Tanaka et al. position of the drug molecule to albumin have not been clarified. It is important in the molecular design of a drug to elucidate the binding position, and it paves the way for development of new drugs with little side effects. In the studies of interaction between drugs and water-soluble polymers, equilibrium dialysis was commonly used to study the binding constant (K), the number (n) of binding sites, and nature of binding (hydrophobic or hydrophilic). On the other hand, NMR especially the ratio of the spin-spin relaxation rate (1/T 2 ) of the free drug to that of the bound drug was the most useful parameter to determine the binding position of the drug with serum albumin 2), which is based on the modification of spin-echo method 3) and the Carr-Purcell- Meiboom-Gill (CPMG) method 4). Here the binding position of Bezafibrate to human serum albumin (HSA) was studied by examining NMR relaxation time to clarify the essence of binding microscopically. It is concluded that the hydrophobic aromatic moiety of BZF was the principal binding position to HSA. EXPERIMENTAL BZF was of special reagent grade from Sigma, and used without further purification. HSA (mol. wt., ) was from Sigma. Other reagents were commercially available and used without further purification. The procedure of equilibrium dialysis was as follows. Dialysis membrane (0.09 mm in thickness) supplied by Visking Company was boiled four times for 5 min each, and interposed between two parts of a dialysis cell made of polymethylmethacrylate. The HSA solution or phosphate buffer solution (0.1M, ph 7, for the control experiment) was injected into one side of the cell, and the drug solution into the other side, the volume of each side being 0.8 ml. After the cell had been shaken in a thermostat at a given temperature regulated within 0.2 C for 24 h, the absorbance of the drug without HSA was measured on a UV spectrometer (Shimadzu UV-190 spectrometer). Parameters of BZF at ph 7 are as follows: max in nm ( ): 227 (18200). The NMR spectra were measured in deuterium oxide (D 2 O, phosphate buffer, 0.1M, ph 7) on a JEOL GX-400 spectrometer (radio frequency, 400MHz, /2 pulse, 11.1 sec) at 40 C. Isotope effects on binding constant were uncorrected. The spin-lattice relaxation time (T 1 ) was obtained by inversion recovery method (Eq.1): ln(m 0 -M t ) = -t/t 1 + ln(2m 0 ) (1) where t is the interval between and /2 pulses, and M 0 and M t represent equilibrium magnetization at t = 0 and macroscopic magnetization at t, respectively. The spin-spin relaxation time (T 2 ) was determined according to Carr-Purcell-Meiboom-Gill (CPMG) method (Eq.2): ln(m t ) = ln(m 0 )-t/t 2 (2) where t is the time when a free induction decay (FID) is observed after application of /2 pulse, and M t is intensity of a spin echo at t. The pulse delay time (20 s), when the next pulse was applied after observation of the FID, was longer than the relaxation time T 1 by

3 Interaction between Drugs and Biomedical Materials 753 a factor of five or above, as required to avoid saturation. A homo-gated irradiation technique was used to depress the HDO peak in D 2 O. RESULTS AND DISCUSSION 1/r (mol/mol) Binding Constants of BZF to HSA The binding of the drug (BZF) to protein (HSA) was examined by equilibrium dialysis in the temperature range of 20 C to 40 C. Free drug concentration (D f ) and the number of the drug bound to a molecule of HSA (r) were estimated from the concentration of the dialyzed drug. A plot of 1/D f versus 1/r produced a linear relationship, as shown in Fig.1, and satisfied Eq.3 proposed by Klotz 5) : 1/r = 1/(nKD f ) + 1/n (3) where n is the number of binding sites per molecule of HSA, and K represents the binding constant of the drug to HSA. The values of n and K were calculated from intercept and slope of the line. Table 1 summarizes thermodynamic parameters calculated from the linear relationship between ln K and the reciprocal of absolute temperature. For the BZF-HSA system, the free energy changes ( G ) and standard enthalpy changes ( H ) were negative, and standard entropy changes ( S ) were positive. These results indicated that the decrease of energy was due to the hydrophobic interaction between BZF and HSA, and that the increase of entropy was based on the destruction of the iceberg structure induced by the hydrophobic interaction. As shown in Fig.2, the binding constant was independent of ionic strength. The binding constant decreased with the addition of short-chain fatty acid ([Caprylic acid]=1mm, K= M -1 ). These results suggest that the binding of BZF to HSA was due to a hydrophobic mechanism /r (mol/mol) /D f ( 10 3 L/mol) /D f ( 10 3 L/mol) Fig.1 Klotz plots for the binding of BZF to HSA ( M) in 0.1M phosphate buffer (ph 7) at 20 C( ), 30 C( ), 40 C( ). Fig.2 Klotz plots for the binding of BZF to HSA ( M) in phosphate buffer (0.025M;, 0.05M;, 0.1M;, 0.2M;, ph 7) at 30 C.

4 754 M. Tanaka et al. Table 1. Thermodynamic data for the binding of BZF with HSA. a) Temp. ( C) K ( 10 3 M -1 ) G (kj/mol) H (kj/mol) S (J mol -1 K -1 ) a) HSA, M; phosphate buffer, 0.1M, ph=7. Chemical Shift of BZF As is shown in Table 2, both 10-times dilution of BZF and addition of HSA ( M) resulted in slight differences in the chemical shift of BZF. Therefore, it was difficult to determine the binding position from the change in the chemical shift; even the largest shift of 6-CH was as small as ppm. Table 2. Chemical shifts ( ) of BZF a) Cl 6 7 HN 1-CH 3 2-CH 2 3-CH 2 4-CH 5-CH 6-CH 7-CH (s) (t) (t) (d) (d) (d) (d) 1mM BZF mM BZF mM BZF/ M HSA a) External reference, tetramethylsilane; ph=7, 40 C; (s), singlet; (d), doublet; (t), triplet. O 3 2 Spin-Lattice Relaxation Time of BZF Spin-lattice relaxation time (T 1 ) of BZF was independent of the concentrations (1-10mM)(Table 3). The addition of HSA ( M) to BZF (10mM) caused a significant decrease in the relaxation time. It was predicted that the phenyl group of BZF mainly interacted with HSA, since the T 1 values of aromatic protons decreased more than those of aliphatic protons. 5 4 O 1 COOH Table 3. Spin-lattice relaxation time (T 1,s) of BZF a) 1-CH 3 2-CH 2 3-CH 2 4-CH 5-CH 6-CH 7-CH 1mM-BZF mM BZF mM BZF/ M HSA a) ph=7, 40 C.

5 Interaction between Drugs and Biomedical Materials 755 Spin-spin Relaxation Rate Spin-spin relaxation time (T 2 ) of BZF was measured by CPMG method (Fig.3). Spinspin relaxation rates (1/T 2 ) of BZF were almost independent of the concentrations (1-10 mm) (Table 4). The addition of HSA ( M) to BZF (10 mm) caused a significant increase in the relaxation rate. Owing to the lack of HSA on chemical shift, slow exchange between free and bound states was reported to cause the superposed narrow and broad peaks in the NMR spectrum. In this work, however, rapid exchange between both sites was understood, since one peak was observed as the weighted average of two states. In a slow exchange system, T 2 measurement by CPMG method was found to be difficult because of modulation action by repeated irradiation of the pulse. In this work, a series of echoes measured by CPMG method decayed according to Eq. 2 without modulation. Therefore, the exchange between free and bound states in our system was suggested to be rapid, similar to the case reported by Jardetzky 6). The spin-spin relaxation rate of drug bound to albumin, (1/T 2 ) b, was calculated according to the equation proposed by Jardetzky: 1/T 2 = (1-B)(1/T 2 ) f + B(1/T 2 ) b (4) where (1/T 2 ) f is the spin-spin relaxation rate of the free drug, and B is the proportion of the drug bound to albumin. The B value can be calculated from the binding constant (K) and the number of binding sites (n) obtained by equilibrium dialysis. Fehske et al 7). reported that the binding sites of two hydrophobic drugs, warfarin and diazepam, were tryptophan and tyrosine residues on HSA, respectively, and that a maximum 10.7 units of 18 units of tyrosine in HSA were modified. Therefore, the largest number of hydrophobic binding sites on HSA was assumed to be 12, which consisted of 1 unit tryptophan and 11 units tyrosine. The values of (1/T 2 ) b were calculated according to Eq.4. t(sec) HDO Fig. 3 Spin-spin relaxation traces obtained by the CPMG method for the protons of BZF.

6 756 M. Tanaka et al. Table 4. Spin-spin relaxation rates (1/T 2,s -1 ) of BZF a) n=5 n=12 Peak (1/T 2 ) f1 (s -1 ) (1/T 2 ) f2 (s -1 ) 1/T 2 (s -1 ) (1/T 2 ) b ( 10 3 s -1 ) (1/T 2 ) b /(1/T 2 ) f2 ( 10 3 ) (1/T 2 ) b ( 10 3 s -1 ) However, the discussion of the binding position on the bases of (1/T 2 ) b values was not reasonable, since the relaxation rates of the respective protons of BZF, (1/T 2 ) f2, were different from each other. The ratio (1/T 2 ) b /(1/T 2 ) f2 has a significant meaning for determination of the binding position, since the sequence of the ratio was independent of the arbitrary n-values (5 or 12), although the absolute values of the ratio depended on these n-values. As can be seen from Table IV, the 6-CH proton had the largest value of (1/T 2 ) b /(1/T 2 ) f2, followed by the 7-CH proton. It was therefore concluded that the binding position of BZF to HSA was at the hydrophobic aromatic moiety. In Fig.4, the contour plot of the (1/T 2 ) b /(1/T 2 ) f2 values was illustrated for visual representation of the binding position. Fig. 4 Contour plot of the ratio of the spin-spin relaxation rate of bound BZF to free BZF. (1/T 2 ) b /(1/T 2 ) f2 ( 10 3 ) 1-CH CH CH CH CH CH CH a) (1/T 2 ) f1, free (1mM) observed; (1/T 2 ) f2, free (10mM) observed; 1/T 2, overall observed (10mM BZF/ M HSA); (1/T 2 ) b BZF bound to HSA calculated; n, number of binding sites on HSA. References 1. M. Hayasaka, Y. Tomioka, S. Ishiwata, M. Mizugaki, Iryo Yakugaku, 28(2), 101 (in Japanese) (2002). 2. M. Tanaka, Y. Asahi, S. Masuda, and K. Minagawa, Chem. Pharm. Bull., 46(5), 817(1998). 3. E. L. Hahn, Phys. Rev., 80, 580(1950). 4. S. Meiboom and D. Gill, Rev. Sci. Instrum., 29, 688(1958). 5. I. M. Klotz, F. M. Walker, and R. B. Pivan, J. Am. Chem. Soc., 68, 1486(1946). 6. O. Jardetzky, Adv. Chem. Phys., 7, 499(1964). 7. K. J. Fehske, W. E. Muller, and U. Wollert. Biochim. Biophys. Acta, 577, 346(1979); idem, Mol. Pharmacol., 16, 778(1979).

Asian Journal of Chemistry; Vol. 25, No. 4 (2013),

Asian Journal of Chemistry; Vol. 25, No. 4 (213), 214-218 http://dx.doi.org/1.14233/ajchem.213.13346 Observation of Triplet Traces Obtained with Inversion Recovery Method in Both Residual Water- and H