Langmuir 2000, 16, 1557-1565
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Effect of the Presence of β-Cyclodextrin on the Solution Behavior of Procaine Hydrochloride. Spectroscopic and Thermodynamic Studies C. Merino,† E. Junquera,† J. Jime´nez-Barbero,‡ and E. Aicart*,† Departamento de Quı´mica Fı´sica I, Facultad de Ciencias Quı´micas, Universidad Complutense de Madrid, 28040 Madrid, Spain, and Departamento de Quı´mica Orga´ nica Biolo´ gica, Instituto de Quı´mica Orga´ nica, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain Received May 11, 1999. In Final Form: October 25, 1999 The effect of the addition of β-cyclodextrin (CD) to the aqueous solutions of the local anesthetic drug, procaine hydrochloride, has been fully investigated by means of spectroscopic (UV-vis, steady-sate fluorescence, and NMR) and thermodynamic (density and speed of sound) studies. The global picture of the results indicates that procaine hydrochloride penetrates the CD cavity by the wider ring, -NH2 group end first, allowing up to the aromatic ring of the drug inside the cavity, with a 1:1 stoichiometry. A new model has been proposed to determine binding constants from UV-vis spectra, when the addition of CD provokes a wavelength shift instead of an absorbance increase. The association constant, obtained from both emission fluorescence and UV-vis data, ranges from 400 to 200 M-1, on going from 15 to 40 °C. A linear decrease of the affinity of the cyclodextrin by the drug with temperature drives the enthalpy (∆H° ) -19 ( 5 kJ mol-1) and the entropy (∆S° ) -15 ( 7 J K-1 mol-1) changes of the binding process to negative values. These values indicate that the encapsulation of procaine hydrochloride by β-cyclodextrin is an exothermic and enthalpy governed process, with a balance between van der Waals contacts, hydrophobic effect, and solvent reorganization being mainly responsible for the overall stability of the complex. The thermodynamic study has shown that in the reorganization of water molecules after the association of the CD and the drug, four to five water molecules are expelled from the CD cavity and four to five water molecules are removed from the hydration shell of procaine.
Introduction Molecular complexation with artificial receptors has become an increasingly used method in many technological and research fields.1-5 Besides, highly specific biological processes make extensive use of molecular complexation, with noncovalent interactions playing an important role.6,7 Many researchers working in the field of molecular recognition processes have focused their studies on a number of therapeutic molecules, whose bioavailability is often threatened by problems such as limited solubility or stability, and a series of undesirable adverse effects. Among the different methods proposed to improve the development of drug delivery systems, molecular complexation with cyclodextrins (CDs) has been generally accepted as one of the most efficient.1-3 Natural cyclodextrins, which have become nowadays a whole new family * Corresponding author. Phone number: 34-91-3944208. Fax number: 34-91-3944135. E-mail:
[email protected]. http:// www-quifi.quim.ucm.es/ea. † Universidad Complutense de Madrid. ‡ Instituto de Quı´mica Orga ´ nica, CSIC. (1) (a) Szejtli, J.; Osa, T. Comprehensive Supramolecular Chemistry, Vol. 3, Cyclodextrins; Elsevier: Oxford, 1996. (b) Szejtli, J. Industrial Applications of Cyclodextrins, Vol. 3; Academic Press: London, 1984. (c) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, 1998. (2) (a) D’Souza, V. T.; Lipkowitz, K. B. Chem. Rev. 1998, 98, 1741. (b) Mulski, M. J.; Connors, K. A. Supramol. Chem. 1995, 4, 271. (c) Connors, K. A. Chem. Rev. 1997, 97, 1325. (3) Thompson, D. O. Crit. Rev. Therapeutic Drug Carrier Systems 1997, 14, 1-104. (4) Luzzi, L.; Palmieri, A. Biomedical Applications of Microencapsulation; Lim, F., Ed.; CRC Press: Boca Raton, FL, 1984. (5) Ducheˆne, D.; Wouessidjewe, D. J. Coord. Chem. 1992, 27, 223. (6) Chadwick. D.; Widdows, K. Host-Guest Molecular Interactions: From Chemistry to Biology; Ciba Foundation Symposium 158; Wiley: Chichester, 1991. (7) Dugas, H. Bioorganic Chemistry: A Chemical Approach to Enzyme Action; Springer-Verlag: New York, 1989.
of pharmaceutical excipients, are torus-shaped cyclic oligosaccharides, containing six (R-CD), seven (β-CD), or eight (γ-CD) R-1,4-linked glucopyranose units, with a relatively hydrophobic central cavity and hydrophilic entrances. Among them, β-CD and its derivatives are dimensionwise the most interesting for drug complexation. The sequestration of a hydrophobic molecule, or some part of it, inside the cavity results in the formation of noncovalent inclusion complexes,1,2 with physical, chemical, and biological properties that may be dramatically different from those of either the parent drug or the cyclodextrin. van der Waals contacts, hydrophobic and solvent effects, hydrogen bonding, and solvent reorganization have been proposed8,9 as the driving forces for the encapsulation process. The rational design of formulations which take advantage of cyclodextrin complexation requires a good understanding of the encapsulation equilibrium. Structural information, such as the stoichiometry and geometry of the complex, and thermodynamic information, such as the association constant and the change on the enthalpy, entropy, and heat capacity of binding, are necessary to draw a complete picture of the interaction CD/Drug. It is known1-5 that host-guest formation in aqueous solution should occur with extensive desolvation and solvation of the host, guest, and complexed species. Consequently, characterization of the hydration properties in these systems should provide improved insight into the role of solute-solute and solute-solvent interactions, associated with the formation of these complexes. A complete understanding of such hydration phenomena requires (8) (a) Bergeron, R. J.; Channing, M. A.; McGovern, K. A. J. Am. Chem. Soc. 1978, 100, 2878. (b) Cromwell, W. C.; Bystro¨m, K.; Eftink, M. R. J. Phys. Chem. 1985, 89, 326. (c) Martin-Davies, D.; Savage, J. R. J. Chem. Soc., Perkin Trans. 2 1994, 1525. (9) Junquera, E.; Aicart, E. J. Phys. Chem. B 1997, 101, 7163.
10.1021/la9905766 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/04/1999
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determination of partial molar volumes and compressibilities, which to our knowledge are scarce measurements in the case of CD/Drug systems. The investigation of all these aspects is the main objective of this work, focused on the analysis of the inclusion complex formed by the anesthetic drug procaine hydrochloride and β-cyclodextrin. Procaine and its derivatives make up an important class of synthetic drugs of the therapeutic family of local anesthetics, whose structures resemble natural compounds actively participating in nerve impulse transmission. It is believed that the cationic form of the drug, which seems to be the active principle, joins the Na+ channels on the nerve membrane, thus blocking the initiation and transmission of nerve impulses.10 However, local anesthetics often show a short duration of action and adverse side effects, such as cardiac and neurological toxicity, accompanied sometimes by allergic reactions. It is then expected that the formulation of procaine hydrochloride as a microencapsulate with a cyclodextrin may show a better bioavailability, with all or some of these undesirable effects masked or abolished. This work reports a complete thermodynamic (speed of sound, density, and partial molar related properties) and spectroscopic (UV-vis, steady-state fluorescence, and NMR) analysis of the effect of the presence of β-cyclodextrin on the properties of aqueous solutions of procaine hydrochloride. We believe that the results of these studies will show the convenience of characterizing the CD/Drug complexes, not only from a pharmacological (pharmacokinetic and in vivo experiments) point of view but also from a physicochemical point of view, to improve our understanding of the CD/Drug interactions and, in a final step, the quality of drug action. Experimental Section Materials. Procaine hydrochloride (ProcHCl), and β-cyclodextrin (β-CD) were purchased from Sigma. Both substances, with purities higher than 99%, were used without further purification. A thermogravimetric analysis (TG) revealed that β-cyclodextrin consists of 11% water mass content, which was considered in the calculations of solute concentrations. All the solutions were freshly prepared with distilled and deionized water (taken from a Millipore Super-Q System, with a conductivity lower than 18 µΩ-1 cm-1). The homogeneity of the initial solutions was assured by sonicating them for 3 h in an ultrasonic bath. UV-vis Measurements. The UV-vis spectra were recorded at 15, 25, and 40 °C with a Varian Cary 5G double beam UVvis-NIR spectrophotometer, from 190 to 400 nm with 1 nm intervals. The equipment is connected to a Pentium PC via a IEEE-bus interface. Data acquisition and analysis of UV-vis spectra were performed with software supported by the manufacturer. Two 10 mm stoppered rectangular silica UV cells (sample and reference cells) were placed in a stirred cuvette holder, whose temperature was kept constant within (0.05 °C with a recirculating ethylene glycol-water circuit. The scan rate was selected in all cases as 300 nm/min. On the experiments with the Drug/water binary systems, the ProcHCl concentration was varied from 0.0 to 11.1 mM, while for the β-CD/Drug/water ternary systems, the drug concentration was kept constant at 5.0 × 10-5 M and β-CD concentration was varied from 0 to ∼8 mM, both in the sample and in the reference cells. Fluorescence Measurements. Steady-state fluorescence experiments were carried out with a Perkin-Elmer LS-50B luminiscence spectrometer. The equipment was connected to a PC-486 computer via a RS-232C interface. Data acquisition and analysis of fluorescence spectra were performed with the Fluorescence Data Manager Software supported by Perkin(10) (a) Bowman, W. C.; Rand, M. J. Textbook of Pharmacology; Blackwell Sci. Pubs., University Press: Cambridge, 1990. (b) Avendan˜o, C. Introduccio´ n a la Quı´mica Farmace´ utica; McGraw-Hill-Interamericana: Madrid, 1993. (c) Alcolea, M. Spectrosc. Lett. 1997, 30, 975.
Merino et al. Elmer. Details of the experimental procedure were fully described earlier.11 A 10 mm stoppered rectangular silica cell was placed in a stirred cuvette holder whose temperature was kept constant within (0.01 °C. Both the excitation and emission slits were fixed at 5 nm, the excitation wavelength was set at 305 nm, and the emission spectra were collected from 315 to 500 nm. In all the measurements the scan rate was selected at 240 nm/min. The titrations were made at several temperatures from 15 to 40 °C, at a constant drug concentration of ∼5 × 10-6 M, varying the β-CD concentration from 0 to ∼7 mM. NMR Measurements and Docking Study. NMR experiments were performed on a Varian Unity 500 spectrometer at 26.0 ( 0.1 °C. Monodimensional 500 MHz 1H NMR spectra were recorded for the aqueous solutions of (i) procaine hydrochloride at 61.4 mM, (ii) β-CD at 14.0 mM, and (iii) a series of samples of β-CD at a constant concentration of 14.0 mM with variable drug concentration. A capillary tube of hexadeuterated acetone was used to provide the deuterium lock. The Watergate sequence12 was used to suppress the water signal on these spectra. A bidimensional nuclear Overhauser effect (NOE) experiment13 was run for the sample with the highest CD/Drug ratio, corresponding to ∼80% of complex formation. The NOESYWatergate spectrum was performed with a data matrix of 512 × 2K to digitize a spectral width of 4000 Hz. Thirty-two scans were used with a relaxation delay of 1.5 s. A mixing time of 600 ms was used. The docking calculation of the complex β-CD/ ProcHCl was performed in a Silicon Graphics workstation with the MM3* force field as implemented in MACROMODEL v4.514 and the GB/SA solvent model for water.15 Density and Speed of Sound Measurements. The speed of sound was measured by a pulse-echo-overlap (PEO) technique, operating with broadband pulses at 2.25 MHz. Calibration of the distance between the transducer and the reflector was made from the speed of sound in pure water at 298.15 K.16 The reproducibility of the speed of sound measurements is (0.02 m s-1. The density of the solutions was measured using a flow densimeter, sensitive to 2 ppm or better. The period of the vibrating tube of the densimeter, calibrated with vacuum and water, was obtained as an average over 104 periods. The temperature control system keeps the temperature constant within (1 mK. The details of the apparatuses and the experimental procedures for both techniques have been fully described in previous papers.17 Measurements of the speed of sound and the density of aqueous solutions of procaine hydrochloride were performed, at 25 °C, as a function of drug concentration (i) in the absence of cyclodextrin and (ii) in the presence of a constant concentration of β-CD at 14.73 mM.
Results and Discussion Procaine Hydrochloride Aqueous Solutions. Procaine hydrochloride, an ester of the p-aminobenzoic acid, may suffer hydrolysis in aqueous media. Besides, its cationic form (ProcH+) is in equilibrium with its nonionized form (Proc), as follows Ka
H2N-Ph-COO-(CH2)2-NH(C2H5)2+Cl- {\} ProcHCl H2N-Ph-COO-(CH2)2-N(C2H5)2 + H+Cl- (1) Proc (11) Junquera, E.; Aicart E. J. Inclusion Phenom. 1997, 29, 119. (12) Piotto, M.; Saudek, V.; Sklenar, V. J. Biomol. NMR 1992, 2, 661. (13) Neuhaus, D.; Williamson, M. P. The Nuclear Overhauser Effect in Structural and Conformational Analysis; VCH: New York, 1989. (14) Mohamadi, F.; Richards, N. G. I.; Guida, W. C.; Liskamp, R.; Canfield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440. (15) Still, W. C.; Temczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am. Chem. Soc. 1990, 112, 6127. (16) Kroebel, W.; Mahrt, K. H. Acustica 1976, 35, 154. (17) (a) Tardajos, G.; Dı´az-Pen˜a, M.; Aicart, E. J. Chem. Thermodyn. 1986, 18, 683. (b) Pastor, O.; Junquera, E.; Aicart, E. Langmuir 1998, 14, 2950.
Cyclodextrin in Procaine Hydrochloride
Figure 1. UV-vis spectra of aqueous solutions of ProcHCl at different concentrations, at 25 °C: 1, 0.645 × 10-5 M; 2, 1.48 × 10-5 M; 3, 2.56 × 10-5 M; 4, 3.96 × 10-5 M; 5, 5.51 × 10-5 M; 6, 7.07 × 10-5 M; 7, 8.55 × 10-5 M; 8, 9.90 × 10-5 M; 9, 11.1 × 10-5 M.
where Ka is the equilibrium constant. With the aim of confirming that the hydrolysis of the ester does not occur in aqueous media, the pH of the aqueous solutions of ProcHCl was measured as a function of drug concentration at 25 °C with an experimental computerized procedure widely described previously.9 From these experimental pH data, the Ka for procaine hydrochloride was determined as a fit parameter of a NLR method.9 A value of 5 × 10-10 at 25 °C (pKa ) 9.3) has been obtained, in agreement with literature data,18 thus revealing that in an aqueous solution of procaine hydrochloride with the concentration ranges used in all the studies reported herein, (i) the ester is not hydrolyzed and (ii) equilibrium 1 is almost totally shifted toward the ionized form of the drug (ProcH+), with a negligible contribution of the nonionized form (Proc). The UV-vis spectra of the drug in aqueous solution were collected at different drug concentrations at 15, 25, and 40 °C. Figure 1 shows, as an example, the experiment performed at 25 °C. It can be observed that the spectra show three peaks centered at λmax ) 194, 221, and 291 nm, with absorbance values increasing with drug concentration, following a typical Lambert-Beer behavior. Only three temperatures were checked in the UV experiments since a negligible dependence with temperature was observed on the UV spectra of the drug solutions. From the linear regression of the values of the absorbances at the λmax as a function of drug concentration, the molar absorption coefficients, , were determined as 22 770 ( 70, 9240 ( 2, and 19 780 ( 40 M-1 cm-1 at 194, 221, and 291 nm, respectively. The third peak, corresponding to the π f π* transition (k band), at 291 nm, is the one used to follow the emission fluorescence spectra, as a result of the excitation on the tail of the absorption band in order to avoid an undesirable inner filter effect. The curve labeled 0 in Figure 2 shows the emission fluorescence spectrum of a 4.99 × 10-6 M aqueous solution of procaine hydrochloride, characterized by a band centered at 358 nm with a low fluorescence intensity (∼92 au), as expected for an apolar molecule within a polar environment. (18) Shuang, S.; Guo, S.; Pan, J.; Li, L.; Cai, M. Anal. Lett. 1998, 31, 879.
Langmuir, Vol. 16, No. 4, 2000 1559
Figure 2. Emission fluorescence spectra of an aqueous solution of ProcHCl at constant concentration (4.992 × 10-6 M) at 25 °C, in the absence and presence of different concentrations of β-CD: 0, 0 M; 1, 1.05 × 10-4 M; 2, 2.08 × 10-4 M; 3, 4.11 × 10-4 M; 4, 6.09 × 10-4 M; 5, 9.89 × 10-4 M; 6, 1.61 × 10-3 M; 7, 2.42 × 10-3 M; 8, 3.33 × 10-3 M; 9, 4.52 × 10-3 M; 10, 5.51 × 10-3 M; 11, 6.33 × 10-3 M; 12, 7.04 × 10-3 M.
The thermodynamic study has been carried on at 25 °C, by measuring the speed of sound, u, and the density, d, of aqueous solutions of procaine hydrochloride. Two interesting quantities can be obtained from these data: the isoentropic compressibility, κs, and the hydration number, nh, which are related19,20 with the molality of the solution, m, and the isoentropic compressibility of pure water, κs,o, as follows
κs ) 1/du2 nh )
(
(2)
)
nw κs 1ns κs,o
(3)
where nw and ns are the number of moles of water and solute, respectively. All the quantities (u, F, κs, and nh) vary linearly with concentration, the fit coefficients being reported in Table 1. When ProcHCl is dissolved in water, both the cationic form of the drug and the Cl- anion are surrounded by water molecules. The hydrophobic hydration dominates the solvation of the aromatic and alkyl chain parts of the molecule, while the polar and charged groups interact with water via electrostatic forces. Since the nh value for Cl- anion is known to be ∼0,19 the data in Table 1 indicate that the procaine cation (ProcH+) is surrounded by 14 water molecules in its hydration shell, this value remaining almost unchangeable with the concentration. Apparent molar volumes, Vφ, and isoentropic compressibilities, Ks,φ, are known to provide useful information (19) Bockris J. O’H.; Reddy, A. K. N. Modern Electrochemistry; Plenum Publishing Co.: New York, 1977. (20) Moulik, S. P.; Gupta, S. Can. J. Chem. 1989, 67, 356. (21) (a) Zana. R. Surfactant Solutions: New Methods of Investigation; Marcel Dekker, Inc.: New York, 1987. (b) Millero, F. J. Chem. Rev. 1971, 71, 147. (c) Millero, F. J.; Lo Surdo, A.; Shin, C. J. Phys. Chem. 1978, 82, 784. (d) Millero, F. J.; Laferriere, A. L.; Chetirkin, P. V. J. Phys. Chem. 1977, 81, 1737. (e) Kharakoz, D. P. J. Phys. Chem. 1991, 95, 5634. (f) Chalikian, T. V.; Sarvazyan, A. P.; Breslauer, K. J. Biophys. Chem. 1994, 51, 89. (g) Chalikian, T. V.; Sarvazyan, A. P.; Breslauer, K. J. J. Phys. Chem. 1993, 97, 13017.
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Table 1. Fit Coefficients of the Plots of u, d, Ks, and nh as a Function of the Molarity for the β-Cyclodextrin and the Procaine Hydrochloride in the Absence and in the Presence of β-CD, at 25 °C fit coefficients
u (m s-1)
d (10-3 kg m-3)
κs (10-12 Pa-1)
nh
a0 a1 std dev
1496.78 185.69 0.03
β-CD 0.997053 0.419743 6 × 10-6
447.67 -298.3 0.02
39.5 -338 0.2
a0 a1 std dev
1496.67 161.57 0.04
ProcHCl 0.997039 0.047208 5 × 10-6
447.74 -116.7 0.02
13.9 7.2 0.1
a0 a1 a2 std dev
1500.36 141.50
442.85 -99.1
12.8 -1.3
0.04
0.3
ProcHCl + β-CDa 1.003150 0.033436 0.198254 0.07 5 × 10-6
a
Data for procaine hydrochloride in the presence of 14.73 mM of β-CD.
about the degree and nature of the solute hydration.21 These quantities can be calculated from density or isoentropic compressibility data by
M 1000(d - do) Vφ ) d mddo Ks,φ ) Vφκs +
1000(κs - κs,o) mdo
(4)
(5)
where M is the molar mass of the solute, m is the molality of the solution, and d and κs and do and κs,o are the densities and the isoentropic compressibilities of the solution and the solvent, respectively. From these values, the apparent o , have molar quantities at infinite dilution, Voφ and Ks,φ 21b,d as follows been determined
Vφ - Svm1/2 ) Voφ + bvm
(6)
o bκsm Ks,φ - Sκsm1/2 ) Ks,φ
(7)
where Sv and Sκs are the Debye-Hu¨ckel limiting slopes, available from the literature.21b-d In the case of nonionic solutes, i.e., β-CD, the term containing the Debye-Hu¨ckel limiting slope on eqs 6 and 7 disappears. Consequently, plots of (Vφ - Svm1/2) and (Ks,φ - Sκsm1/2) as a function of m (Figure 3) should give straight lines, whose intercepts and slopes represent the apparent molar properties at o ) and the deviation paraminfinite dilution (Voφ and Ks,φ eters (bv and bκs) from the Debye-Hu¨ckel limiting law (DHLL), respectively. Table 2 reports these coefficients for the aqueous solution of ProcHCl at 25 °C. The signs of the slopes bv and bκs are determined by the nature of the interaction between solute species. The overlap of hydrophobic hydration cospheres of apolar groups drives to negative values for both parameters (bi < 0), while positive values (bi > 0) are characteristic of overlapping of the hydration cospheres of polar or charged groups. Data in Table 2 and Figure 3 show that the volumetric properties slightly decreases with drug concentration, indicating that the hydrophobic interaction between solute molecules is slightly dominant. On the other hand, the o , obtained in the infinite dilution quantities Voφ and Ks,φ range where the solute-solute interaction disappears, provide information about the solute-solvent interactions.
Figure 3. Plot of Vφ - Svm1/2 and Ks,φ - Sκsm1/2 of aqueous solutions of ProcHCl as a function of concentration, in the absence of β-CD (squares) and in the presence of a constant [β-CD] ) 14.73 mM (circles), at 25 °C. o Table 2 reports the values obtained for Voφ and Ks,φ for ProcHCl in water, in reasonable agreement with available literature data.22 The value of Voφ consists of two contributions, the intrinsic volume of the solute molecule and o depends just on the that of its hydration shell, while Ks,φ hydration shell, since the solute intrinsic compressibility is assumed to be zero. The term “hydration shell” is referred to those water molecules which, due to the presence of the solute, show a different behavior with respect to those molecules belonging to the bulk. Several models can be found in the literature 21e,23,24 to estimate the solute apparent molar properties. By use of the additive model21e,23 and literature data for the group contributions,21c,e,23 a value of Voφ ) 230.4 cm3 mol-1 has been obtained, in very good agreement with the experimental data reported herein. Higher discrepancies have o ) -15.4 × 10-15 m3 been found on the estimation of Ks,φ -1 -1 mol Pa due to the lack of data of the group contributions for this property with respect to the experimental results. β-CD/Procaine Hydrochloride Aqueous Solutions. When a cyclodextrin solution is added to a ProcHCl aqueous solution, the formation of the inclusion complex CD/Drug takes place, following the equilibrium
KCD:Drug
β-CD + Drug {\} β-CD/Drug
(8)
A complete characterization of this supramolecular entity requires the determination of several magnitudes: the stoichiometry, the binding constant (KCD/Drug), the geom(22) (a) Iqbal, M.; Verrall, R. E. Can. J. Chem. 1989, 67, 727. (b) Iqbal, M.; Jamal, M. A.; Ahmed, M.; Ahmed, B. Can. J. Chem. 1994, 72, 1076. (c) Matsuki, H.; Hashimoto, S.; Yamanaka, M. Langmuir 1994, 10, 1882. (23) (a) Traube, J. Samml. Chem. Chemischtechn. Vortra¨ ge 1899, 4, 7. (b) Cabani, S.; Gianni, P.; Mollica, V.; Lepori, L. J. Solution Chem. 1981, 10, 563. (c) Gianni, P.; Lepori, L. J. Solution Chem. 1996, 25, 1. (d) Hoiland, H.; Vikingstad, E. Acta Chem. Scand., Ser. A 1976, 30, 692. (24) (a) Terasawa, S.; Isuki, H.; Arakawa, S. J. Phys. Chem. 1975, 79, 2345. (b) Edward, J. T.; Farrell, P. G. Can. J. Chem. 1975, 53, 2965. (c) Cohn, E. J.; Edsall, J. T. Proteins, Amino acids and Peptides; Reinhold: New York, 1943. (d) Lee, B. J. Phys. Chem. 1983, 87, 112. (e) Kharakoz, D. P. J. Solution Chem. 1992, 21, 569.
Cyclodextrin in Procaine Hydrochloride
Langmuir, Vol. 16, No. 4, 2000 1561
Table 2. Fit Coefficients of the Plots of VO-Svm1/2 and Ks,O-SKsm1/2 as a Function of the Molality for β-CD,a Procaine Hydrochloride in the Absence and in the Presence of β-CD, and for the Complex β-CD/Procaine Hydrochloride, at 25 °C substances
Voφ (10-6 m3 mol-1)
bv (10-6 m3 mol-2 kg)
std dev
o Ks,φ (10-15 m3 mol-1 Pa-1)
bκs (10-15 m3 mol-2 kg Pa-1)
std dev
β-CDa ProcHCl ProcHCl + β-CDb β-CD/ProcHCl
710.0 226.8c 239.4 243.0
962.6 -18.4 -222.9 -88.1
0.7 0.1 0.3 0.4
-6.7 -10.5d 4.8 1.7
3620 -82.6 -100.6 634.2
1.9 0.9 2.6 4.6
a For nonionic solutes, S and S are zero in eqs 6 and 7. b Data for procaine hydrochloride in the presence of 14.73 mM β-CD. c V ° ) v κs φ 226.6 × 10-6 m3 mol-1 (ref 22a); Vφ° ) 225.8 × 10-6 m3 mol-1 (ref 22b); Vφ° ) 225.5 × 10-6 m3 mol-1 (ref 22c). d Ks,φ° ) -13.1 × 10-15 m3 mol-1 Pa-1 (ref 22a).
Table 3. Values of the Association Constant (KCD:Drug) and the Proportionality Constant Ratio (kCD:Drug/kDrug) at Different λem and Different Temperatures, Obtained from Steady-State Fluorescence Measurements for the System β-CD + Procaine Hydrochloridea
Figure 4. Plot of I/Io at λem ) 350 nm (9, from fluorescence titration) and ∆λ at A ) 0.6 (O, from UV-vis titration) as a function of β-CD concentration at 25 °C.
etry, and thermodynamic information such as ∆H°, ∆S°, and ∆Cop, and the apparent molar quantities for the inclusion process. Figure 2 shows the effect of the addition of β-CD on the fluorescence emission spectra of an aqueous solution of ProcHCl at a constant concentration of 4.99 × 10-6 M at 25 °C, as an example. As can be seen in the figure, the increase of the β-CD concentration results in clear emission intensity enhancements. It is well-known that the intensification of luminescent processes of lumiphores partially or totally encapsulated by the CD cavity is a result of the better protection from quenching and other processes occurring in the bulk solvent. The experimental I/Io values (I and Io being the fluorescence intensity of the solution in the presence and absence of cyclodextrin), plotted as a function of β-CD concentration in Figure 4 (left axis), can be fitted to the well-known fluorescence binding isotherm25
1 + (kCD/Drug/kDrug)KCD/Drug[CD] I ) Io 1 + KCD/Drug[CD]
(9)
by using a NLR analysis based on a Marquardt algorithm. The ratio kCD/Drug/kDrug, where ki represents the proportionality constants connecting the intensities and concentrations, and the association constant of the CD/Drug inclusion complex KCD/Drug, can be obtained as fitting parameters. Table 3 reports the results obtained at three different emission wavelengths λem, at all the temperatures. It can be observed in the table that the values of (25) Connors, K. A. Binding Constants. The Measurement of Molecular Complex Stability; John Wiley: New York, 1987.
[Drug] (10-6 M)
T (°C)
λem (nm)
KCD:Drugb (M-1)
kCD/Drug/kDrugb
f
std dev (10-2)
4.999
15
5.000
20
4.992
25
5.003
30
5.003
35
5.003
40
340 350 360 340 350 360 340 350 360 340 350 360 340 350 360 340 350 360
428 436 441 368 395 400 330 326 335 285 300 289 252 269 265 212 228 228
17.7 13.3 10.7 18.6 13.3 10.6 17.7 12.6 9.9 17.9 12.3 10.0 16.0 11.3 9.1 16.8 11.2 9.1
0.69 0.70 0.70 0.73 0.74 0.74 0.70 0.70 0.70 0.67 0.68 0.67 0.64 0.65 0.65 0.60 0.62 0.62
0.16 0.13 0.10 0.23 0.16 0.13 0.15 0.16 0.10 0.13 0.15 0.08 0.10 0.09 0.08 0.12 0.09 0.08
a λ exc ) 305 nm, λem ranges from 315 to 500 nm, ex/em slit widths ) 5/5 nm, scan rate ) 240 nm/min. b Uncertainties less than 8%.
KCD/Drug are not affected by the λem chosen to analyze the I/Io data and that a proper range of the saturation degree, f, has been covered in all the inclusion processes studied, the goodness of the method and the accuracy of the results being thus guaranteed.26 This fact confirms as well the 1:1 stoichiometry of the β-CD/ProcHCl complex, initially assumed in eq 8. Both the 1:1 stoichiometry and the values of the binding constants were corroborated with the results of the UVvis study of the complex. Figure 5 shows the UV spectra of an aqueous solution of procaine hydrochloride at a constant concentration of 5.0 × 10-5 M in the absence and in the presence of different β-CD concentrations, at 25 °C, as an example. The formation of the inclusion complex provokes a clear bathochromic shift of the three peaks observed in the spectrum of the pure drug. The fact that several isosbestic points appear indicates the presence of a 1:1 complex, thus confirming the fluorescence results. Regarding the determination of the binding constant from these UV data, the method commonly used in the literature25 is the nonlinear regression of the ∆A/b values vs [CD] at a fixed wavelength (normally the maximum), ∆A and b being the increase in the absorbance provoked by the presence of cyclodextrin and the dimension of the cuvette, respectively. However, it is important to state that this method, although indistinctly used in most of the reported UV studies, is only correct when the effect caused by the presence of the cyclodextrin is just the (26) Deranleau, J. Am. Chem. Soc. 1969, 91, 4044.
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Figure 5. UV-vis spectra of an aqueous solution of ProcHCl at constant concentration (5.002 × 10-5 M) at 25 °C, in the absence and presence of different concentrations of β-CD: 0, 0 M; 1, 0.010 × 10-3 M; 2, 0.295 × 10-3 M; 3, 0.672 × 10-3 M; 4, 1.37 × 10-3 M; 5, 2.51 × 10-3 M; 6, 3.77 × 10-3 M; 7, 5.24 × 10-3 M; 8, 6.70 × 10-3 M; 9, 8.00 × 10-3 M. Table 4. Values of the Association Constant (KCD:Drug) and the Maximum Wavelength Shift (∆λmax) at Two Absorbances (A) and Different Temperatures, Obtained from UV-vis Measurements for the System β-CD + Procaine Hydrochloride [Drug] (10-5 M)
T (°C)
5.009
15
5.002
25
5.009
40
a
A
KCD/Druga (M-1)
∆λmaxa (nm)
f
std dev (10-2)
0.6 0.7 0.6 0.7 0.6 0.7
294 347 202 239 152 177
9.7 9.0 10.7 10.1 9.5 9.1
0.71 0.74 0.63 0.67 0.55 0.59
0.04 0.05 0.08 0.08 0.07 0.08
Uncertainties less than 25%.
increase in the absorbance of a given peak, without any wavelength shift. This is not the case of the spectra reported herein, and with the aim of determining the KCD/Drug even when either bathochromic or hipsochromic shifts are observed, we propose in this work a new model. The change of experimental wavelength, ∆λ, observed at a fixed A as a consequence of the formation of the complex must be proportional to the saturation degree, f
∆λ ) ∆λmax f )
∆λmaxKCD/Drug[CD] 1 + KCD/Drug[CD]
(10)
where ∆λmax is the maximum value of these shifts at an hypothetical 100% of saturation degree. From a NLR analysis (based on a Marquardt algorithm) of the experimental ∆λ data, at a given A, as a function of cyclodextrin concentration (see Figure 4, right axis), the ∆λmax and the KCD/Drug values can be obtained as fitting parameters. Table 4 reports the results obtained at the three temperatures studied and at two chosen A values. The uncertainties on the K values obtained from the UV data are expected to be higher than those obtained from the fluorescence results, since the variation observed on the experimental property due to the presence of increasing CD concentrations (i.e. ∆λ and I/Io, respectively) is notoriously lower in
Figure 6. van’t Hoff plots for the associations of β-CD and ProcHCl, from both fluorescence (0) and UV-vis (9) results.
the case of UV-vis. Despite that, the values obtained for the binding constants with the UV-vis method may be considered in reasonably good agreement with those obtained from fluorescence, considering the well-known discrepancies found in the association constants determined with different techniques.25 Furthermore, the scarce data found for this system in the literature are in very good concordance with the results reported herein.18,27 It can be observed in Tables 3 and 4 that as long as the temperature increases, the affinity of the cyclodextrin for the drug decreases. Figure 6 shows the van’t Hoff plots with the fluorescence and the UV-vis data. If ∆Cop ) 0, the experimental R ln K values fit the well-known28 linear equation
R ln K) -∆H°/T + ∆S°
(11)
and ∆H° and ∆S°, which are temperature independent, can be estimated from the slope and intercept of the fit, respectively. Although only three temperatures were studied in the UV experiments, it can be remarked that both van’t Hoff lines drive to a value of -19 ( 5 kJ mol-1 for ∆H° and comparable values (within their uncertainties) for ∆S° -15 ( 7 J mol-1 K-1 from fluorescence and -21 ( 9 J mol-1 K-1 from UV-vis, all in reasonable good agreement with literature.18,27 The inclusion process of procaine by β-CD is exothermic and enthalpy driven (|∆H° | > T|∆S°|), as usually found9,28 for associations between small guest molecules and an apolar cavity in water. A combination of hydrophobic effect (∆H° ∼ 0, ∆S° > 0), van der Waals forces (∆H° < 0, ∆S° < 0), and solvent reorganization could account for such a thermodynamic pattern. The 1D 1H NMR spectra of β-CD at constant concentration in the presence of increasing amounts of drug are useful to verify the formation of the inclusion complex between the CD and the drug. Moreover, the induced (27) Takisawa, N.; Shirahama, K.; Tanaka, I. Colloid Polym. Sci. 1993, 271, 499. (28) (a) Inoue, Y.; Liu, Y.; Tong, L.-H.; Shen, B.-J.; Jin, D.-S. J. Am. Chem. Soc. 1993, 115, 10637. (b) Rekharsky, M. V.; Schwarz, F. P.; Tewari, Y. B.; Goldberg, R. N.; Tanaka, M.; Yamashoji, Y. J. Phys. Chem. 1994, 98, 4098. (c) Junquera, E.; Martı´n-Pastor, M.; Aicart, E. J. Org. Chem. 1998, 63, 4349. (d) Stauffer, D. A.; Barrans, Jr. R. E.; Dougherty, D. A. J. Org. Chem. 1990, 55, 2762.
Cyclodextrin in Procaine Hydrochloride
Figure 7. 1H NMR signals for the H-3 and H-5 protons of β-CD free and bound with ProcHCl at 26 °C.
chemical shifts observed upon inclusion of the guest helped us also to establish an approximate geometry for the complex. The observed effects on the chemical shifts and line broadening indicate that the interaction is fast in the chemical shift NMR time scale. The maximum upfield shifts found in the H-3 (0.12 ppm) and H-5 (0.21 ppm) β-CD protons, located inside the cavity,1,2 for around 80% of complex formation (see Figure 7), indicate the inclusion of ProcHCl. The resonances of the other CD protons showed minor variations. With regard to the drug, the major variations were observed for the aromatic meta protons (0.10 ppm), while smaller changes were detected for those protons located in the position ortho to the COO group (0.03 ppm). Although the larger shift induced in the meta protons with respect to the ortho aromatic protons, together with the larger upfield shift observed for the H-5 proton with respect to the H-3 proton of β-CD, already provided enough evidence for the inclusion of ProcHCl through the wider entrance of the CD torus, with the -NH2 group end first, NOE experiments were carried out to unambiguously demonstrate this hypothesis. Obviously, since NMR parameters are essentially time-averaged, the information that is possible to deduce from NOE experiments corresponds to the time-averaged conformation in solution. At 26 °C, NOESY cross peaks are positive at 500 MHz. Apart of the expected intrarresidue H-1/H-2 and interresidue H-1i/H-4i+1 cross peaks for the β-CD moiety, medium size intermolecular cross peaks were observed between H-3 and H-5 protons of the β-CD and the meta aromatic protons of ProcHCl. No other intermolecular peaks were detected. H-H distances were estimated from the volumes of meta-aromatic/ortho-aromatic cross peaks in relation to the volumes of the intermolecular metaaromatic/H-3, H-5 cross peaks. The estimated average distances were 3.0 ( 0.1 Å. The existence of these NOEs
Langmuir, Vol. 16, No. 4, 2000 1563
is also in agreement with the observed chemical shift major changes in both H-3 and H-5 protons of the CD moiety and on the meta-aromatic protons of the drug. Molecular mechanics calculations were performed to get a three-dimensional structure of the β-CD/ProcHCl complex in aqueous solution. The experimental intermolecular distances were included as constraints to get the complex. In a first step, the drug was manually docked within the CD cavity through its wider entrance, and then a minimization was carried out with the MM3* force field and the GB/SA solvent model for water. After 2000 conjugate gradient iterations, the constraints were removed and the complex was further minimized. Figure 8 shows views of the complex. It is evident that ProcHCl stays within the CD torus and that van der Waals contacts between the aromatic ring and the nonpolar faces of the glucose units take place. Proton-proton distances between 2.2 and 4.4 Å are observed between the meta-aromatic protons of the drug and H-3 and H-5 of the CD. No hydrogen bonds are evident from the obtained structure, since the amino group is more than 4 Å from the CD hydroxyl groups. Besides, no additional hydrogen bond contacts are evident for the carbonyl group, which remains outside of the cavity. However, the partial embedding of the aromatic ring may explain the observed minor shift of the aromatic protons located ortho to the -COO group. The aliphatic chains and the protonated nitrogen remain completely outside of the torus. All the thermodynamic quantities determined for the binary Drug/water system have been also obtained for the ternary β-CD/Drug/water system at 25 °C. Equations 3, 6, and 7 can be used as well for the ternary system, but eqs 4 and 5 must contain an additional term21d which takes into account the cyclodextrin, as follows
Vφ )
M 1000(d - do) mCDMCD(d - do) (12) d mddo mddo
Ks,φ ) Vφκs +
1000(κs - κs,o) mCDMCD(κs - κs,o) + (13) mdo mdo
where do and κs,o are now referred to the values of the mixed solvent, i.e., the β-CD/water solution. Figure 3 shows the plot of (Vφ - Svm1/2) and (Ks,φ - Sκsm1/2) as a function of molality for the ternary system. Tables 1 and 2 report the coefficients of the fits. It can be noticed that (i) the hydration number of ProcHCl, although remaining basically constant with drug concentration in the presence of β-CD, decreases from 14 (in the absence of cyclodextrin) to 13 water molecules and (ii) the apparent molar quantities at infinite dilution for the ternary system, Voφ o and Ks,φ , higher than those obtained for the binary system, decrease linearly with drug concentration. The transfer functions, defined as the transfer value of the solute quantity ∆Yoφ from the water solvent to the mixed β-CD/water solvent, give information about the relative changes that occur in solute solvation upon complexation. At infinite dilution, they are given by the following expression:
∆Yoφ(wfCD/w) ) Yoφ(CD/w) - Yoφ(w)
(14)
o ) 15 × 10-15 Values of ∆Voφ ) 12.6 cm3 mol-1 and ∆Ks,φ -1 -1 mol Pa have been obtained for procaine in the presence of β-CD. These values stand for the global contribution of ProcHCl and β-CD/ProcHCl species to the transfer quantities. For that reason, it would be interesting
m3
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Figure 8. Views of the complex β-CD/ProcHCl.
The encapsulation process may be explained as an overall result of the reorganization of solute and solvent molecules after the association. Scheme 1 shows the solvation, desolvation, and solvent reorganization processes associated with the formation of the CD/Drug inclusion complex in aqueous media. It means that when
Scheme 1 β-CD‚pH2O + Drug‚mH2O a β-CD‚(p - p′)H2O/Drug‚(m - m′)H2O + (p′ + m′)H2O
Figure 9. Plot of Vφ - Svm1/2 and Ks,φ - Sκsm1/2 of an aqueous solution of β-CD/ProcHCl inclusion complex as a function of concentration, at 25 °C.
to estimate the apparent molar properties of the inclusion complex alone, given that the stoichiometry and the association constant are known from the spectroscopic studies. On this calculation, we may assume that the contributions of the free and complexed procaine to the apparent molar quantity are additive,29 as follows
ProcHCl enters the CD cavity, some of its hydration water molecules (m′) pass from the hydration shell to the bulk, meanwhile p′ water molecules are expelled from inside the CD cavity to the bulk, as well. Considering that the NMR and docking results show that only the -NH2 and the aromatic groups of the drug are inside the cavity, the ∆Voφ value consists of the following terms
∆Voφ ) ∆Vp′ + Vw + m′ × 18.07 - Voφ
(16)
where XDrug and XCD/Drug, and Yφ,Drug and Yφ,CD/Drug are the molar fractions and the apparent molar quantities of the free and complexed drug, respectively. Thus, from the value of KCD/Drug ()330 M-1 at 25 °C) and the apparent molar quantities previously obtained for the procaine in the absence and in the presence of cyclodextrin, the values of Yφ,CD/Drug can be obtained. Figure 9 shows the plot of Vφ - Svm1/2 and Ks,φ - Sκsm1/2 for the complex β-CD/ProcHCl as a function of solute concentration. The extrapolations o ) 243.0 cm3 mol-1 to infinite dilution drive to Vφ,CD:Drug o -15 3 -1 and Ks,φ,CD:Drug ) 1.7 × 10 m mol Pa-1, the ∆Voφ and o values being now 16.2 cm3 mol-1 and 12.2 × 10-15 ∆Ks,φ 3 m mol-1 Pa-1.
where Vw is the van der Waals volume of these groups which remain unsolvated when encapsulated by the CD, ∆Vp′ is the volume change due to the expulsion of p′ water molecules from inside the cavity to the bulk, and Voφ is the volume occupied by these groups outside the cavity in aqueous media. We have estimated these contributions as follows: (i) Voφ has been obtained (86.1 cm3 mol-1) using the additive model and the group contributions data reported in the literature for these groups.23b,c (ii) Vw has been calculated (53.9 cm3 mol-1) from the group contributions to the van der Waals volume data,23c using the group increments for molecular properties reported by Bondi.30 (iii) ∆Vp′ has been estimated (-31 cm3 mol-1) through the volume of the CD cavity (157 cm3 mol-1)2a and the number of water molecules housed therein (7),2a independently of the number of water molecules expelled from the cavity when the drug is encapsulated. Nevertheless, it is admitted31 that ∆Vp′ is due to the expulsion of the four to five molecules residing within the hydrophobic core of the CD cavity; the remaining water molecules located in the
(29) (a) Hoiland, H.; Ringseth, J. A.; Vikingstad, E. J. Solution Chem. 1978, 7, 515. (b) Wilson, L. D.; Verrall, R. E. J. Phys. Chem. B 1997, 101, 9270. (c) Wilson, L. D.; Verrall, R. E. J. Phys. Chem. B 1998, 102, 480.
(30) Bondi, A. Physical Properties of Molecular Crystals, Liquids, and Glasses; John Wiley & Sons: New York, 1968. (31) Linder, K.; Saenger, W. Angew. Chem., Int. Ed. Engl. 1978, 17, 694.
Yφ ) XDrugYφ,Drug + XCD/DrugYφ,CD/Drug
(15)
Cyclodextrin in Procaine Hydrochloride
hydrophilic annular region are believed to be similar to the bulk water. Thus, considering the value of 16.2 cm3 mol-1 experimentally determined in this work for ∆Voφ, and using eq 16, we have estimated as four to five the number of water molecules (m′) that ProcHCl leaves free upon binding the cyclodextrin. Conclusions This work reports a complete physicochemical characterization of the inclusion complex formed by β-CD and an anesthetic drug, the cationic form of the procaine hydrochloride, β-CD/ProcHCl. It has been found that the drug penetrates the CD cavity by the wider ring, -NH2 group end first, allowing up to the aromatic ring of the drug inside the cavity, with a 1:1 stoichiometry and a binding constant of 330 M-1 at 25 °C. The association process is exothermic and enthalpy driven, with the
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contribution of van der Waals contacts, the hydrophobic effect, and the reorganization of solvent molecules to the overall stability of the complex. The thermodynamic study has shown that after the encapsulation of the drug by the CD cavity, there is a neat change of one water molecule on the hydration of the drug, in concordance with the volume and isoentropic compressibility transfer quantities obtained herein. This low change has been explained in terms of a balance among the transfer volumes of four to five water molecules being expelled from the CD cavity and four to five water molecules being removed from the hydration shell of procaine, when it is encapsulated. Acknowledgment. This research was supported by the Ministerio de Educacio´n y Cultura of Spain through Project DGES PB98-0755. LA9905766
