Quantitative Prediction of the Suppression of Drug-Induced Hemolysis

Drug-Induced Hemolysis by Cyclodextrins from Surface ... Both the capabilities of CyDs for hemolysis suppression and surface tension elevation are in ...
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Langmuir 1999, 15, 594-599

Quantitative Prediction of the Suppression of Drug-Induced Hemolysis by Cyclodextrins from Surface Tension Data Noriaki Funasaki,* Mariko Ohigashi, Sakae Hada, and Saburo Neya Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Received June 2, 1998. In Final Form: November 10, 1998 The suppression of hemolysis induced with 0.7 mmol dm-3 chlorpromazine hydrochloride (CPZ) or 15 mmol dm-3 propantheline bromide (PB) by R-, β-, and γ-cyclodextrins (CyDs) is measured as a function of CyD concentration and is correlated with the surface tension of its solution determined at 310 K. The surface tension data allow us to estimate the 1:1 and 2:1 binding constants of CPZ with CyDs as well as the dimerization constant of CPZ. The 2:1 binding constant of CPZ with γ-CyD is larger than the 1:1 binding constant, whereas the converse result is observed for the PB-γ-CyD system. This cooperative binding of CPZ to γ-CyD would be ascribed to a higher tendency of CPZ to form dimer than that of PB. Both the capabilities of CyDs for hemolysis suppression and surface tension elevation are in the order β-CyD > γ-CyD > R-CyD for 0.7 mmol dm-3 CPZ, whereas those capabilities are in the order β-CyD ≈ γ-CyD > R-CyD for 15 mmol dm-3 PB. The bitter taste reduction and surface tension elevation for a 1.5 mmol dm-3 PB solution were both in the order β-CyD > γ-CyD > R-CyD. This discrepancy for PB is ascribed to the effects of self-association of PB and the 2:1 binding at the high PB concentration. The suppression of CPZ- or PB-induced hemolysis for β- and γ-CyDs can be quantitatively predicted from the observed surface tension data, regardless of the kind and concentration of CyD, but it is not the case for R-CyD. This exception is ascribed to weaker competitive binding of these drugs to R-CyD than that of membrane phospholipid.

Introduction Cyclodextrins (CyDs) are doughnut-shaped macrocyclic glucose polymers, attached by R-(1,4) linkages. Cyclohexaamylose (R-CyD), cycloheptaamylose (β-CyD), and cyclooctaamylose (γ-CyD) are best characterized among the CyDs. The inner diameters of the cavities of R-, β-, and γ-CyDs are approximately 0.45, 0.7, and 0.85 nm, respectively. Roughly speaking, the cavity sizes of R-, β-, and γ-CyDs best accommodate benzene, naphthalene, and anthracene, respectively.1-3 Since the interior of the doughnut, lined with CH groups, provides a relatively hydrophobic environment, hydrophobic guest molecules can be entrapped therein. Generally, the equilibrium binding constants for the 1:1 stoichiometry of guest and CyD have been determined rather accurately, but those values for other stoichiometries are less accurate.1-4 Furthermore, to our knowledge, the effects of selfassociation of guest molecules on the binding constants have been neglected for most reports. Cyclodextrins have widespread applications in pharmaceuticals, cosmetics, and foods, since they are practically nontoxic. CyDs have the hemolytic effect at high concentrations,5,6 since they can extract cholesterol and phospholipid from erythrocyte membranes.1,3,5,7 At low * Corresponding author: Phone +81-75-595-4663; Fax +81-75595-4762; E-mail [email protected]. (1) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1988; Chapters 1 and 3. (2) Bender, M. L.; Komiyama, M. Cyclodextrin Chemistry; SpringerVerlag: Berlin, 1978; Chapters 2 and 3. (3) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344. (4) Connors, K. A. Chem. Rev. 1997, 97, 1325. (5) Fro¨mming, K.-H.; Szejtli, J. Cyclodextrins in Pharmacy; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1994; Chapters 3, 6, and 10. (6) Ohtani, Y.; Irie, T.; Uekama, K.; Fukunaga, K.; Pitha, J. Eur. J. Biochem. 1989, 186, 17. (7) Ishikawa, S.; Neya, S.; Funasaki, N. J. Phys. Chem. 1998, 102, 2502.

Figure 1. Chemical structures of (a) chlorpromazine hydrochloride (CPZ) and (b) propantheline bromide (PB).

CyD concentrations, however, they can alleviate the hemolytic effects and the bitter taste intensities of drugs.1,5,8 Generally, both the desired pharmacological effects and the unwanted side effects are elicited only by the uncomplexed drug molecules.5 Chlorpromazine hydrochloride (CPZ) has the phenothiazine ring (Figure 1) and self-associates micellarly in aqueous solutions.9-11 This tranquilizer is a typical membrane-active drug12 and causes hemolysis by intravenous injection. This CPZ-induced hemolysis is suppressed by the addition of CyDs.13 The 1:1 binding (8) Funasaki, N.; Uemura, Y.; Hada, S.; Neya, S. J. Phys. Chem. 1996, 100, 16298. (9) Funasaki, N.; Hada, S.; Paiement, J. J. Phys. Chem. 1991, 95, 4131. (10) Attwood, D.; Florence, A. T. Surfactant Systems; Chapman and Hall: London, 1983; Chapter 4. (11) Funasaki, N. Adv. Colloid Interface Sci. 1993, 43, 87. Funasaki, N.; Hada, S.; Neya, S. Trends Phys. Chem. 1997, 6, 253. (12) Seeman, P. Pharmacol. Rev. 1972, 24, 583.

10.1021/la980643g CCC: $18.00 © 1999 American Chemical Society Published on Web 12/24/1998

Quantitative Prediction of Hemolysis Suppression

constants of CPZ with CyDs at 298 K were reported,14,15 although the effects of self-association of CPZ on these constants were not taken into consideration. Proton NMR and induced circular dichroism (CD) studies provided evidence for the 2:1 stoichiometry of CPZ and γ-CyD,16 but its binding constant has not yet been determined. Propantheline bromide (PB) is a bitter anti-acetylcholine drug. This drug self-associates nonmicellarly.17 The bitter taste intensity of a 1.5 mmol dm-3 PB solution is remarkably reduced by the addition of CyDs. The extent of reduction by the CyDs can be quantitatively predicted from the observed surface tension values of their solutions at 310 K.8 At this low PB concentration, only the dimerization of PB influences the binding of PB with CyDs.8 Drugs inducing strong hemolysis are generally surfaceactive, whereas CyDs are extremely surface-inactive. Therefore we might expect that the strength of hemolysis of an aqueous solution containing a drug and cyclodextrin is determined by the surface tension value for the solution, regardless of the kind and concentration of CyD.8 Then, we can predict the extent of hemolysis from the observed surface tension value alone. The first purpose of this work is to demonstrate that we might quantitatively predict the extent of suppression of drug-induced hemolysis by R-, β-, and γ-CyDs from observed surface tensions at 310 K, as has already been reported for the reduction of bitter taste of PB.8 The second purpose is to determine the 1:1, 1:2, and 2:1 binding constants of drug and CyD, with taking into consideration the self-association of CPZ9 and PB,17 by the surface tension method.8,18,19

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Figure 2. Hemolytic effects of R-, β-, and γ-CyDs on human erythrocytes in 10 mmol dm-3 phosphate buffer (pH 7.4) containing 140.5 mmol dm-3 NaCl at 310 K: 0, β-CyD (this work); curve a, R-CyD (literature);6 curve b, β-CyD (literature);6 curve c, γ-CyD (literature).6

Experimental Section Materials. Commercial samples of CPZ and PB (Sigma) were used without further purification. R-, β-, and γ-CyDs were purchased from Nakalai Tesque Co. (Kyoto). Their water contents were determined from their dry weights. All inorganic salts used (NaBr, NaCl, NaH2PO4‚2H2O, and Na2HPO4‚12H2O) were of analytical grade from Wako Pure Chemicals Co. (Osaka). Ionexchanged water was used after double distillation. Methods. All experiments were carried out at 310 K in the presence of 140.5 mmol dm-3 NaCl (for CPZ) or NaBr (for PB) and 10 mmol dm-3 phosphate buffer (pH 7.4). The surface tension was measured by the Wilhelmy method. The equilibrium surface tension was used for further analysis. Human blood was collected from healthy donors, with 0.47% sodium citrate as an anticoagulant. Erythrocytes, separated by centrifugation at 1000g for 10 min, were washed three times with 10 mmol dm-3 isotonic phosphate buffer (pH 7.4) and then suspended in the buffer solution to give a hematocrit value of 10%. To 4 cm3 of the buffer solution containing CPZ or PB was added 0.4 cm3 of the erythrocyte suspension. The mixture was incubated for 30 min at 310 K and then centrifuged at 1000g for 30 min. The percent hemolysis was determined from the absorbance at 543 nm due to hemoglobin in the supernatant, and 100% hemolysis is the 543 nm absorbance of erythrocyte solution hemolyzed completely with distilled water.20 (13) Uekama, K.; Irie, T.; Sunada, M.; Otagiri, M.; Iwasaki, K.; Okano, Y.; Miyata, T.; Kase, Y. J. Pharm. Pharmacol. 1981, 33, 707. (14) Otagiri, M.; Uekama, K.; Ikeda, K. Chem. Pharm. Bull. 1975, 23, 188. (15) Uekama, K.; Irie, T. Chem. Lett. 1978, 1109. (16) Takamura, K.; Inoue, S.; Kusu, F. Chem. Lett. 1983, 233. (17) Funasaki, N.; Uemura, Y.; Hada, S.; Neya, S. Langmuir 1996, 12, 2214. (18) Funasaki, N.; Yodo, H.; Hada, S.; Neya, S. Bull. Chem. Soc. Jpn. 1992, 65, 1323, and references therein. (19) Dharmawardana, U. R.; Christian, S. D.; Tucker, E. E.; Taylor, R. W.; Scamehorn, J. F. Langmuir 1993, 9, 2258. (20) Fujii, T.; Sato, T.; Tamura, A.; Wakatsuki, M.; Kanaho, Y. Biochem. Pharmacol. 1979, 28, 613.

Figure 3. (a) Hemolysis by aqueous CPZ solutions and (b) the surface tension of its solutions in 10 mmol dm-3 phosphate buffer (pH 7.4) containing 140.5 mmol dm-3 NaCl at 310 K. The solid line in panel b is calculated from eqs 5 and 6 with the values of A, B, C, and k2 given in the text.

Results and Discussion Hemolysis by Cyclodextrins. First, we investigated the hemolytic activity of β-CyD. As Figure 2 shows, β-CyD begins to hemolyze at 3 mmol dm-3 and hemolyzes almost completely at 9 mmol dm-3. Our results are in excellent agreement with the literature data,6 carried out under similar conditions. Figure 2 also shows that the hemolytic activity of CyD is strong in the order β-CyD > R-CyD > γ-CyD. CyD forms pores in the erythrocyte membrane by the extraction of its lipid: R-CyD extracts phospholipid mainly, and β-CyD and γ-CyD extract cholesterol mainly.6 Owing to the hemolytic effects of CyDs, therefore, their parenteral administration is not approved in many countries.5 Suppression of CPZ-Induced Hemolysis and Elevation of Surface Tension of CPZ Solutions by CyD. The percent hemolysis by CPZ also increases with increasing CPZ concentration (Figure 3a) and reaches 100% at 0.7 mmol dm-3. CPZ penetrates the erythrocyte membrane and results in hemolysis, owing to making it fragile as an exogenous membrane component. The surface tension of an aqueous CPZ solution decreases with increasing concentration (Figure 3b). This decrease is ascribed to the adsorption of CPZ to the airwater interface. We did not carry out experiments of hemolysis and surface tensions at higher CPZ concentrations. At a higher concentration CPZ must form its micelle. The critical micelle concentration (cmc) of CPZ in a 154

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Figure 4. Effects of R-CyD (O), β-CyD (0), and γ-CyD (4) on (a) the hemolysis and (b) the surface tension of a 0.7 mmol dm-3 CPZ solution in 10 mmol dm-3 phosphate buffer (pH 7.4) containing 140.5 mmol dm-3 NaCl at 310 K. The solid lines in panel a are calculated from eq 17 with the observed surface tension data (panel b). The solid lines in panel b are calculated from eqs 6 and 15 with binding constant data (Table 1).

Figure 5. (a) Hemolysis by aqueous PB solutions and (b) the surface tension of its solutions in 10 mmol dm-3 phosphate buffer (pH 7.4) containing 140.5 mmol dm-3 NaBr at 310 K. The solid line in panel b is calculated from eqs 5 and 7 with the values of A, B, a, b, c, and k2 given in the text.

mmol dm-3 sodium chloride solution is 4.5 mmol dm-3 at 298 K, and CPZ also forms dimer under the same conditions.9 As Figure 3a shows, CPZ completely hemolyzes at 0.7 mmol dm-3. The hemolysis induced by 0.7 mmol dm-3 CPZ is suppressed by the addition of either β- or γ-CyD but remains unchanged by the addition of R-CyD (Figure 4a). The suppression of hemolytic activity by CyD is in the order β-CyD > γ-CyD > R-CyD. The surface tension of the 0.7 mmol dm-3 CPZ solution is increased by the addition of these cyclodextrins, in the same order of CyDs (Figure 4b). These changes in hemolysis and surface tension will be mainly due to the reduction of free CPZ concentration induced by the binding of CPZ with CyD. This interpretation might allow us to predict that CPZ does not bind to R-CyD. This prediction, however, is inconsistent with experimental results obtained for the binding of CPZ and R-CyD by various methods.1,15 Suppression of PB-Induced Hemolysis and Elevation of Surface Tension of PB Solutions by CyD. To confirm the above discrepancy for CPZ, we carried out the same experiments for PB. As Figure 5a shows, PB completely hemolyzes at 15 mmol dm-3. The surface tension of an aqueous PB solution decreases with in-

Funasaki et al.

Figure 6. Effects of R-CyD (O), β-CyD (0), and γ-CyD (4) on (a) the hemolysis and (b) the surface tension of a 15 mmol dm-3 PB solution in 10 mmol dm-3 phosphate buffer (pH 7.4) containing 140.5 mmol dm-3 NaBr at 310 K. The solid lines in panel a are calculated from the observed surface tension data (panel b) by using eq 17. The solid lines in panel b are calculated by using binding constant data (Table 1) from eqs 8 and 15.

creasing concentration and levels off around 13 mmol dm-3. This concentration is the cmc under the present conditions. Furthermore, we investigated the effects of CyDs on the hemolysis (Figure 6a) by a 15 mmol dm-3 PB solution. The suppression of PB-induced hemolysis by β-CyD is close to that by γ-CyD. R-CyD does not suppress the hemolysis, as was observed with CPZ (Figure 4a). We have already reported that the reduction of the bitter taste intensity of 1.5 mmol dm-3 PB is in the order β-CyD > γ-CyD > R-CyD.8 This inconsistency in the suppressions of hemolysis and bitter taste by CyDs will be ascribed to the difference between the concentrations of PB used in these experiments. As Figure 6a shows, γ-CyD again increases the percent hemolysis above 20 mmol dm-3 or higher concentrations. This increase may be due to free γ-CyD molecules. Figure 6b shows the effects of CyDs on the surface tension of a 15 mmol dm-3 PB solution. The surface tension increases gradually at low CyD concentrations and rather abruptly at higher concentrations. This increasing pattern is converse, as compared to that for a 1.5 mmol dm-3 PB solution (Figure 7b in ref 8). The power of surface tension elevation by CyDs at 15 mmol dm-3 PB is in the order β-CyD ≈ γ-CyD > R-CyD (Figure 6b), whereas that at 1.5 mmol dm-3 PB is in the order β-CyD > γ-CyD > R-CyD.8 This result also demonstrates the importance of the PB concentration. Estimation of Binding Constants from Surface Tension Data. We have developed a surface tension method for determining the binding constant for the amphiphile-CyD system18 and applied it to a surfactant18 and PB.8 Figure 7 shows a schematic model for a number of equilibria in the drug-CyD system. Many drugs selfassociate stepwise in water owing to hydrophobic interactions10,11 and may form 1:1, 1:2, and 2:1 complexes with CyD.1,4,8,18 In the absence of CyD and erythrocytes, the surface tension, γ, of an aqueous drug solution will be a unique function of the molarity, [P], of drug in the monomer state:

γ ) f{[P]}

(1)

The molarity of dimer is calculated from the dimerization constant, k2:

Quantitative Prediction of Hemolysis Suppression

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We employed the observed surface tension value at Cp ) 0 for γw and a value of k2 ) 13.4 dm3 mol-1 estimated at 310 K by surface tension measurements.8 The parameters in eqs 5 and 6 were determined to be best fit to the observed surface tension data shown in Figure 5b by a nonlinear least-squares method: a ) 3.21, b ) 15.4, c ) 0.441, A ) 17.78, and B ) 1.653. The theoretical surface tension is shown by the solid line in Figure 5b. The molarity of 1:1 complex, PD, is written by using the binding constant, K1, of 1:1 complexation as4,18,21,22 Figure 7. Schematic interrelation among the equilibria of surface adsorption, self-association, CyD inclusion, binding, and extraction for the drug-CyD system.

[P2] ) k2[P]2

(2)

The molarity of i-mer may be calculated from i

[Pi] ) [P] exp(-i∆G°/RT) 2/3

-i∆G°/RT ) -ai + bi

+ ci

(3)

∑3 i[P]i exp(ai - bi2/3 - ci4/3)

(5)

From GFC data, the aggregation parameters for CPZ9 were determined to be k2 ) 124 dm3 mol-1, a ) 27.8, b ) 55.2, and c ) 4.02, and those for PB17 were k2 ) 13.4 dm3 mol-1, a ) 7.85, b ) 12.5, and c ) 2.35 at 298 K. The concentrations of trimer or higher multimers of CPZ are negligible at 0.7 mmol dm-3 at 298 K.9 Thus, we assumed that only dimer of CPZ is present at such a low concentration and that the surface tension of aqueous CPZ solutions could be written by the following empirical equation:

(6)

If we know the k2 value in eq 5, we can determine the concentration, [P], of free CPZ as a function of Cp. Then, if we know the values for A, B, and C in eq 6, we can calculate the theoretical surface tension value of an aqueous CPZ solution as a function of Cp. Thus, these adjustable parameters in eqs 5 and 6 were determined to be best fit to 10 observed surface tension data shown in Figure 3b by a nonlinear least-squares method: k2 ) 99 dm3 mol-1, A ) -17.78, B ) 1.653, and C ) -0.381. The best fitting was obtained by minimizing the SS value: N

SS )

∑(γobsd - γcalcd)2

(7)

where N denotes the number of data. The k2 value at 298 K was used as an initial value. The solid line shown in Figure 3b is theoretically calculated from eqs 5 and 6 with these k2, A, B, and C values. For PB we assumed that the surface tension could be expressed by the Szyszkowski equation:

γ ) γw - A exp(1 + B[P])

where [D] denotes the molarity of free CyD. The molarities of 1:2 and 2:1 complexes are also written as

[PD2] ) K2[P][D]2

(10)

[P2D] ) K3[P]2[D]

(11)

(4)

∞

γ ) A + B exp(C[P])

(9)

The total concentration of drug is expressed as 4/3

Here i∆G° denotes the free energy of i-mer formation from i monomers. By gel-filtration chromatography (GFC) we have shown that CPZ in a 154 mmol dm-3 sodium chloride solution9 and PB in a 154 mmol dm-3 sodium bromide solution17 self-associate stepwise at 298 K and that their concentration can be expressed as

CP ) [P] + 2k2[P]2 +

[PD] ) K1[P][D]

(8)

∞

2

CP ) [P] + 2k2[P] +

∑3 i[P]i exp(ai - bi2/3 - ci4/3) +

K1[P][D] + K2[P][D]2 + 2K3[P]2[D] (12) Here the adsorption amount of drug at the air-water interface is neglected. The total concentration of CyD is written as

CD ) K1[P][D] + 2K2[P][D]2 + K3[P]2[D]

(13)

The concentration [D] of free CyD can be obtained from

[D] ) {-K1[P] - K3[P]2 + [(K1[P] + K3[P]2)2 + 8CDK2[P])1/2}/4K2[P] (14) Substitution of eq 14 into eq 12 yields

Cp ) g{[P]}

(15)

Because g is a rather complicated function containing CD, K1, K2, K3, k2, a, b, and c, we do not write it in explicit form. CyD is surface-inactive enough not to change the surface tension of water. Therefore, we presume that all the complexes of drug and CyD are also surface-inactive.8,18 Then eq 1 holds true even in the presence of CyD. The observed surface tension data for CPZ shown in Figure 4b were analyzed, taking into consideration adequate complex species. The theoretical surface tension for a CPZ solution at a given set of CP ) 0.7 mmol dm-3 and CD was obtained as follows. First, we assumed a set of initial values of K1, K2, and K3. From eq 15 we can calculate [P] and subsequently γ from eq 6. Thus, we can determine the best-fit values for K1, K2, and K3. The binding constants thus obtained are shown in Table 1. For R- and β-CyDs, the binding models taking into consideration 1:2 and/or 2:1 complex species did not improve fitting significantly. Comparison among models was judged from (21) We have used the binding constants defined by the molarity of each species, but those values defined by the mole fraction are better thermodynamically (see ref 22). This difference in concentration units influences the absolute values for the standard free energy and entropy of complexation, but we are not concerned with these thermodynamic values in the present work. The standard enthalpy of complexation is independent of these concentration units. (22) Gurney, R. W. Ionic Processes in Solution; McGraw-Hill: New York, 1953; Chapters 5 and 6.

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Table 1. Binding Constants of CPZ and PB with r-, β-, and γ-CyD K1 K2 K3 temp CyD (dm3 mol-1) (dm3 mol-1) (dm3 mol-1) (K) method ref Rβγ-

Rβγa

CPZ 110 200 6200 12000 7900 420 1000

2300

310 298 310 298 298 310 298

STa UV ST UV CDc ST UV

b 15 b 14 14 b 15

310 310 310

ST ST ST

8 8 8

PB 80 3400 230

1200 70

Surface tension. b This work. c Circular dichroism.

the magnitude of SS. The fitting procedure has already been reported in detail.8,18 The 1:1 binding constants of CPZ with R- and β-CyDs were reported at 298 K in the literature,14,15 and those values are larger than ours at 310 K. This decrease in binding constant with increasing temperature is usually observed; i.e., the enthalpy of complexation is negative.23 For γ-CyD, induced CD and NMR data suggested the presence of 2:1 complex,16 but there was no report on the 2:1 binding constant for this system. Our 2:1 binding constant K3 is much larger than K1: the second CPZ molecule cooperatively binds to the 1:1 complex of CPZ and γ-CyD. It is notable that the K1 value for R-CyD is smaller than those of β- and γ-CyDs by 1 order of magnitude or more (Table 1). This result is consistent with the observed hemolysis result (Figure 4a). For PB, we have already determined the binding constants at 310 K, as shown in Table 1.8 Using these values, we calculated theoretical values of the surface tension, shown by the solid lines in Figure 6b. The agreement between theory and experiment is excellent. In particular, it is worth comparing the observed surface tension data on β- and γ-CyDs. β-CyD elevates the surface tension of 1.5 mmol dm-3 PB more remarkably than γ-CyD,8 but these cyclodextrins elevate the surface tension of 15 mmol dm-3 PB by a similar extent (Figure 6b). These results demonstrate that the self-association of PB and the K3 term remarkably influence the elevation of surface tension by CyDs. CPZ is more surface-active than PB (Figures 3b and 5b), the dimerization constant of CPZ is larger than that of PB, and the cmc of CPZ is smaller than that of PB. These results all indicate a larger hydrophobicity of CPZ over PB. This difference in hydrophobicity is consistent with a stronger hemolysis of CPZ over PB (Figures 3a and 5a). This difference is also reflected in the binding behavior. The binding constants of CPZ with R-, β-, and γ-CyDs are larger than those of PB (Table 1). As Table 1 shows, CPZ binds to γ-CyD cooperatively (K3 > K1), whereas PB binds anticooperatively (K3 < K1). Dodecyl maltoside also binds cooperatively to γ-CyD.18 The 2:1 complex of drug and γ-CyD contains the dimeric structure of drug. The larger dimerization constant of CPZ, compared to PB, is one of the driving forces for the cooperative binding of CPZ. The dimer of CPZ is formed by stacking interactions of its phenothiazine ring in water.24 Both homodimers of CPZ (23) Cramer, F.; Saenger, W.; Spatz, H. C. J. Am. Chem. Soc. 1967, 89, 14. (24) Attwood, D.; Waigh, R.; Blundell, R.; Bloor, D.; The´vand, A.; Boitard, E. Dube`s, J.-B.; Tachoire, H. Magn. Reson. Chem. 1994, 32, 468.

and PB in the γ-CyD cavity will have three-dimensional structures in which their tricyclic rings stack in parallel arrangements.8,16 Thus, CPZ can form the 2:1 complex with γ-CyD without significant changes in the threedimensional structure of dimer. This favors the cooperative binding of CPZ to γ-CyD. This implication has to be confirmed further, since no report is available on the threedimensional structure of the PB dimer in water. Prediction of Hemolysis from Surface Tension Data. Unless CyD and its complexes with a hemolytic drug influence erythrocytes, the percent hemolysis by solutions containing drug, CyD, and erythrocytes will be determined only by the concentration of free drug:

percent hemolysis ) h([P])

(16)

The function h will be determined from a combination of eq 5 with the observed hemolysis data (Figure 3a for CPZ and Figure 5a for PB), if the adsorption amount of drug to erythrocytes is neglected. This assumption is justified approximately, if all hemolysis experiments are carried out at a low concentration of erythrocytes. Then a combination of eqs 1 and 16 yields

percent hemolysis ) h{f-1(γ)}

(17)

This equation predicts that the percent hemolysis by solutions containing drug and CyD is a unique function of surface tension, regardless of the drug concentration and the kinds and concentrations of CyD. On the basis of this prediction, we can calculate the percent hemolysis of the solution from the observed surface tension value of a mixed CPZ and CyD solution (Figure 4b) by using eqs 15 and 17. As the solid line in Figure 4a shows, it is in excellent agreement with the observed hemolysis percent for β- and γ-CyDs, but this is not the case for R-CyD. R-CyD binds weakly to CPZ (Table 1) and can bind more strongly to phospholipid in the erythrocyte membrane. Thus it is likely that most of the CPZ molecules added do not actually complex with R-CyD in the presence of erythrocytes. If this is true, R-CyD will not suppress hemolysis induced by CPZ. The solid line in Figure 6a for PB shows the prediction based on eq 15. This prediction holds true for β- and γ-CyDs but not for R-CyD. This exception for R-CyD is also explicable in terms of stronger competitive binding of membrane phospholipid than PB. As Figure 6a shows for the PB-γ-CyD system, the percent hemolysis increases at high γ-CyD concentrations again. This increase will be attributed to the increase in free CyD concentration. At these high concentrations, however, the concentrations of free γ-CyD molecules, calculated from the binding constants shown in Table 1, are not high enough to hemolyze, as compared to the hemolysis data shown in Figure 1. For instance, the concentrations of free PB and free γ-CyD, [P] and [D] at CD ) 30 mmol dm-3, are 2.2 and 19 mmol dm-3, respectively. These concentrations cause 2.7% (Figure 5a) and 3.9% (Figure 2) hemolysis. The summation of these hemolysis percentages is smaller than an observed value of 25%. At such high γ-CyD concentrations, free γ-CyD and PB molecules would cause hemolysis synergistically: the fragile erythrocyte membrane induced by PB is hemolyzed at a lower γ-CyD concentration than the intact membrane. We neglect this effect in our prediction method. Conclusions Surface tension data allow us to determine the binding constants of these drugs with R-, β-, and γ-CyDs at 310

Quantitative Prediction of Hemolysis Suppression

K. To determine these constants, we took into consideration the decrease in monomer concentration of drug owing to its self-association. The effect of the self-association of drug on CyD inclusion is remarkable, particularly at high drug concentrations. For the first time we determined the 2:1 binding constant of CPZ with γ-CyD. This value is larger than the 1:1 constant. This means that the second CPZ molecule binds cooperatively to the 1:1 complex of CPZ and γ-CyD. On the other hand, the second PB molecule binds anticooperatively to the 1:1 complex of PB and γ-CyD. One of the reasons for this difference in PB and CPZ would be an easier dimerization of CPZ in the γ-CyD cavity than that of PB. Recently, chemistry is rapidly extending to the field of biology. Then we need more physicochemical data at 310 K, the body temperature, instead of 298 K. If we used surface tension data at 298 K, those data would not quantitatively correlate with the percent hemolysis, such as shown in Figures 4a and 6a. The suppression of CPZ- and PB-induced hemolysis by β- and γ-CyDs can be quantitatively predicted from the observed surface tension value alone regardless of the kind and concentration of CyD, but R-CyD does not alleviate hemolysis even at its high concentrations. This anomaly is ascribed to a weaker binding capacity of R-CyD for CPZ and PB than for phospholipid in erythrocyte membranes. Regarding to this point, a further study is underway. Such an exception was not observed for the reduction of the bitter taste intensity by CyDs.8 The

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capabilities for suppression of PB-induced hemolysis and for elevation of surface tension are both in the order β-CyD ≈ γ-CyD > R-CyD,8 whereas the capability for reduction of the bitter taste intensity of PB and for elevation of surface tension are in the order β-CyD > γ-CyD > R-CyD. This discrepancy is ascribed to the difference in PB concentration. As a result, the relative amount of free PB molecules is less at high PB concentrations than at low PB concentrations, and the 2:1 complex of PB with γ-CyD contributes more at high PB concentrations than at low PB concentrations. The present results will generally be observed for other amphiphilic drugs. During the research and development of a novel drug, one has often found that it is too toxic to be used as a medicine, albeit pharmacologically effective. If we could reduce its toxicity by the addition of CyD, it might be utilized commercially. Then, it is desirable that we can determine the kind of concentration of CyD on the basis of some physicochemical measurements and fewer hemolysis experiments. Acknowledgment. Thanks are due to Professor Takashi Satoh, Kyoto Pharmaceutical University, for his kind suggestions on hemolysis experiments. We thank Ms. Yuriko Sako for her valuable help with experiments of PB. LA980643G