ISSN 0036-0244, Russian Journal of Physical Chemistry, 2006, Vol. 80, No. 7, pp. 1050–1055. © Pleiades Publishing, Inc., 2006. Published in Russian in Zhurnal Fizicheskoi Khimii, 2006, Vol. 80, No. 7, pp. 1200–1205.
PHYSICAL CHEMISTRY OF SOLUTIONS
Inclusion Complexation of Itraconazole with b- and 2-Hydroxypropyl-b-Cyclodextrins in Aqueous Solutions1 M. I. El-Barghouthia*, N. A. Masouda, J. K. Al-Kafaweina, and A. A. Abdohb a Department
of Chemistry, Hashemite University, P.O. Box 330117, Zarqa 13133, Jordan Pharmaceutical Manufacturing Company, Naor, Jordan *e-mail:
[email protected]
b Jordanian
Received July 4, 2005
Abstract—The inclusion behavior of Itraconazole (Itra) with β-cyclodextrin (β-CD) and 2-hydroxypropyl-βcyclodextrin (HP-β-CD) was investigated by using phase solubility and molecular mechanics techniques. The effects of pH and temperature on complex stability were also explored. The aqueous solubility of Itra was significantly enhanced as CD concentration increased. Itra tends to form 1 : 3 complexes with β- and HP-β-CD at pH ≥ 4 and 1 : 2 at pH 2. Thermodynamic parameters for Itra/HP-β-CD show that the 1 : 1 complex is driven by enthalpy but retarded by entropy changes. In contrast, the formation of 1 : 2 and 1 : 3 complexes is largely favored by entropy due to higher desolvation induced by total enclosure of Itra with two (or three) favorably interacting CD molecules. The inclusion mode of Itra/β-CD complexes was proved by molecular mechanics technique, which provided a powerful means for understanding inclusion interactions and processes. DOI: 10.1134/S0036024406070090
INTRODUCTION Cyclodextrins (CDs) are oligosaccharides of one to four linked α-4-D-glucose monomers. They are torusshaped molecules with hydrophobic cavities and hydrophilic exterior edges due to hydrophilic hydroxyl groups located at both rims of the CD torus. Because of these structural features, CDs can form inclusion complexes with a variety of organic compounds [1–3]. As a consequence of the inclusion process, many physicochemical properties of guest substrates, such as solubility, dissolution rate, stability, and bioavailability, are favorably changed [3]. Information from model studies on the complexation of CDs can provide a reasonable N
picture of the nature of molecular recognition, which is also significant in understanding enzyme-substrate interactions. Several binding forces have been proposed for the inclusion of substrates into CDs, including van der Waals forces, hydrophobic effects, hydrogen bonding, macrocycle relaxation, and the release of energetic water molecules from the cavity [4, 5]. Itraconazole (Itra) is an orally active, broad-spectrum, triazole antifungal agent [6]. K‡1 of Itra was determined to be 4 while, K‡2 was estimated at 1.5–2 [7]. It is practically insoluble in water, thus causing some difficulties in pharmaceutical formulations of the drug. In this work, the utilization of cyclodextrins to enhance the solubility of Itra will be examined.
N
N O O
N
N
N
O Cl
N N
O
Cl (Itra)
The complexation of Itra with 2-Hydroxypropyl-β-CD (HP-β-CD) in aqueous propylene glycol solution has 1 The
text was submitted by the authors in English.
been studied and found to form 1 : 2 complexes at pH 2 [8]. Peeters et al. studied the interactions of Itra with HP-P-CD at different pH and found that Itra formed a higher order complex with HP-β-CD [7].
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However, the thermodynamics of the complexation process has not been investigated. Also, no study regarding the interaction of Itra with β-CD has been carried out. The present work reports the results of an investigation of the solubility of Itra in aqueous β- and HP-β-CDs solutions. The effects of temperature and pH on complex stability in aqueous solutions are also reported. Individual complex formation constants were estimated through rigorous analysis of the measured phase solubility diagrams. Molecular mechanical computations using an MM+ force field have been carried out to explore possible interaction sites between Itra and CDs [9].
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Seq, må (a)
0.04
0.03
1
0.02
0.01 2
EXPERIMENTAL
3
Materials
0
Itra, β- and HP-β-CDs were provided by JPM (Jordanian Pharmaceutical Manufacturing Co., Naor, Jordan). All reagents used were of analytical grade, and doubly distilled deionized water was used throughout.
5
10
15 20 [β-CD], må
Seq, må 3 (b)
Phase Solubility Studies
2
Excess amounts of Itra were added to 100-ml screw cab flasks. Solutions of CD of various concentrations were prepared at specific pH values in 0.1 M phosphate buffers. Constant volumes of the cyclodextrin solutions were then added to the flasks. The solutions were shaken in a thermostated water bath for 48 h and then left for 24 h to settle and reach equilibrium at fixed temperature. The solutions were then filtered using a 0.45 µm membrane filter and analyzed for Itra. The analysis of Itra was carried out by HPLC. The system configuration included a Perkin Elmer Series 200 LC pump fitted with a 100-µL sample loop and a Perkin Elmer 785A UV/Vis detector (260 nm) dedicated to a PE NELSON 1022 integrator. Samples were eluted with a mixture of acetonitrile, methanol and 0.5% aqueous ammonium acetate in a 51 : 14 : 35 volume ratio on an RP 18 Hypersil®ODS column (100 mm × 4.6 mm, 5 µm particle size) using a 1.6-ml/min flow rate.
1
1 2 3 0
100
200 [HP-β-CD], må
Fig. 1. Phase solubility diagram of Itra against (a) β-CD and (b) HP-β-CD obtained in 0.1 M phosphate buffer and 30°ë at pH (1) 2.0, (2) 4.0, and (3) 7.0.
RESULTS AND DISCUSSION Molecular Modeling Molecular mechanics computations were carried out using the Hyperchem® molecular modeling software (Release 6.03; Hypercube, Inc.; Canada). Energy minimizations in a water box with periodic boundary conditions were obtained using an MM+ force field (0.1 kcal/(mol Å) gradient). The initial molecular geometry of β-CD was obtained using X-ray diffraction data [10]. This geometry was optimized again using the MM+ force field. Itra was built up from standard bond lengths and bond angles. The resulting structure was then minimized with MM+ force fields. RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY
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Figures 1a and 1b depict phase solubility diagrams (PSDs) obtained at 30°ë for Itra against β-CD and HP-β-CD concentration at pH 2, 4, and 7.0 at 30°ë. At the studied pH range, the solubility of Itra has been enhanced remarkably reflecting strong host-guest binding. The obtained PSDs were of Ap type, indicating the formation of higher order complexes (SLn complexes) [11]. The PSDs were utilized to obtain estimates of the complex formation constants and the host-guest stoichiometry of soluble SLn complexes based on the following analysis. No. 7
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EL-BARGHOUTHI et al.
Table 1. Estimates of complex formation constants for Itra/β- and HP-β-CDs obtained from nonlinear regression of PSDs in 0.1 M phosphate buffer at 30°C pH 2.0 4.0 7.0
S0 × 105, mM 10 1.5 1.4
K11 , M–1
K12 , M–1
4630 2390 5700
β-CD 346 352 90
K13 , M–1 – 140 50
K11 , M–1
K12 , M–1
K13 , M–1
2050 8170 19500
HP-β-CD 425 186 25
– 4 6
Table 2. Estimated thermodynamic parameters of Itra/HPβ-CDs complexes obtained in 0.1 M phosphate buffer at 25°C; values in parentheses denote confidence limits Equilibrium reaction
Equilibrium constant
∆G°, *, kJ/mol
∆H°, *, kJ/mol
∆S°, J/(mol K)
S(aq) + L(aq) [SL] (aq) SL(aq) + L(aq) [SL2](aq) S(aq) + 2L(aq) [SL2](aq) SL2(aq) + L(aq) [SL3](aq) S(aq) + 3L(aq) [SL3](aq)
K11 K12 β12 K13 β13
–36.1 (1.7) –15.5 (3.3) –51.7 (5.0) –15.2 (2.0) –66.9 (7.0)
–66.4 (3.6) 104.1 (6.9) 37.7 (10.5) –31.9 (1.3) 5.8 (11.8)
–102 (8.9) 401 (6) 299 (14.9) –56 (5.5) 244 (20.4)
Note: Mole fraction standard state.
The equilibrium solubility (Seq) of Itra in aqueous CD solutions of variable concentrations is given by
the individual complex formation constants by minimizing the sum of squares of errors given by
S eq = S 0 + [ SL ] + [ SL 2 ] + … + [ SL n ] = S 0 + K 11 S 0 [ L ] + K 11 K 12 S 0 [ L ] + …
SSQ = Σ(Seq – S eq )2, p
2
+ K 11 K 12 …K 1n S 0 [ L ] = S 0 + K 11 S 0 [ L ] 3
p
(1)
+ β 12 S 0 [ L ] + … + β 1n S 0 [ L ] , 2
3
where S and L denote Itra and CD, respectively; S0 is the solubility at zero CD concentration; [L] is the concentration of free CD molecules; and SLn represent the 1:n-type complex. K1n and β1n stand for the individual and overall SLn formation constants, respectively. The total concentration of CD in solution (Lt) for the SLn system is given by L t = [ L ] + [ SL ] + 2 [ SL 2 ] + … + n [ SL n ] = [ L ] + K 11 S 0 [ L ] + 2K 11 K 12 S 0 [ L ] + … 2
+ 3K 11 K 12 …K 1n S 0 [ L ] = [ L ] + K 11 S 0 [ L ] 3
(2)
+ 2β 12 S 0 [ L ] + … + 3β 1n S 0 [ L ] . 2
3
It can be shown that [L] is given by 1/2
(3) [ L ] = ( – b ± ( b – 4ac ) )/2a. For the SL2 system, a = 2K11K12S0, b = 1 + K11S0, and c = –Lt. While for the SL3 system a = K11K12S0, b = 2K11S0 – 1 and c = Lt + 3S0 – 3Seq. Nonlinear regressions of experimental data corresponding to each PSD were conducted to obtain S0 and 2
(4)
where S eq is the predicted equilibrium solubility of Itra given by Eq. (1). Rigorous analysis of the PSDs yielded the stoichiometries of Itra-CD complexes and estimates of their formation constants, which are listed in Table 1. It was predicted that both β-CD and HP-β-CD form 1 : 1, 1 : 2, and 1 : 3 soluble complexes at pH 7.0. As shown in Table 1, HP-β-CD showed a stronger 1 : 1 formation constant compared with β-CD at pH 7.0. This finding suggests that the hydroxypropyl groups on the surface of HP-β-CD interact with some groups in Itra and this interaction somehow serves the complexation process. Nevertheless, the overall formation constant was higher for the β-CD case. Some studies of other complexes of HP-β-CD point out that, although the hydroxypropyl group often facilitates the formation of inclusion complexes [12], in some cases it may hinder the penetration of a bulky guest molecule into the cavity [13]. The variation of pH led to significant variations in the complex formation constants. As appears from Table 1, K11 of an Itra/HP-β-CD complex generally increases as pH increases due to change from the cationic to the neutral species of Itra. The trend correlated well with the change in Itra solubility (S0), which means that, the higher the solubility of a drug species in water is, the lower the driving force for its inclusion into the CD cavity is [5]. However, this trend was not observed for the overall complex. Also, the trend was
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INCLUSION COMPLEXATION OF ITRACONAZOLE
absent in the Itra/β-CD complex. This is rather unexpected if the driving force for inclusion stems solely form hydrophobic effects. Therefore, it seems that iondipole and dipole-dipole interactions between protonated Itra and CD play a major role in stabilizing complexes in polar solvent. This is in agreement with Muller and Albers’ findings regarding the complexation of dihydropyridine derivatives with HP-β-CD [14]. One important finding relates to the fact that complex stoichiometry may change on changing the pH of the solution. Thus, for (β-CD and HP-β-CD complexes with Itra, when pH changed from 7.0 to 2.0, complex stoichiometry changed from SL3 to SL2. The effect of temperature on complex stability was studied by constructing PSDs for the neutral Itra/HP-β-CD system at pH 7 and different tempera* , ln K 12 *, tures. Figure 2 shows van’t Hoff plots of ln K 11 and ln K *13 (asterisk indicates mole fraction standard state) against 1/T, while Table 2 lists the corresponding thermodynamic parameters for the formed complexes. It is observed that 1 : 1 complex formation with HP-β-CD is largely driven by a favorable enthalpy change but is hindered by an unfavorable entropy change. In contrast, 1 : 2 complex formation is accompanied by a significant entropy change (∆S° = 299 J/(mol K)). This indicates that the 1 : 1 complex is more tightly bound, with a consequent restricted
N
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structure, while 1 : 2 complex formation is accompanied by more desolvation of Itra and, hence, an appreciable favorable entropy change. Similarly, 1 : 3 complex formation is driven by entropy with a slight unfavorable enthalpy change. Molecular Modeling Only the β-CD/Itra complex was studied. Molecular modeling for HP-β-CD complexes was not possible, due to the difficulty of defining the most realistic structure of this CD, which is a mixture of different isomeric forms. The coordinate system used to define the process of complexation for the two possible approaches, A and B, is shown below. Initially, the position of β-CD was fixed while the guest approached along the x-axis toward the wider rim of CD (or the opposite side of the guest approaching the narrow rim of CD). A reference atom was defined in the guest molecule near the center of mass (labeled with an asterisk in the scheme below). It was set initially at an x-coordinate of –20 Å and was moved through the host wider cavity along the x-axis to +20 Å in 1 Å steps. The structures generated at each step were optimized, while keeping the CD structure fixed. The structures obtained at the minima of the energy surface were further minimized, without restriction to either Itra or CD.
z
N
N O
* N
O
N
N N
N
O Cl
x
O
y
Cl (Approach Ä) N
z
N N
N N
N
N
O
N *
x
O O Cl
O
y Cl (Approach Ç)
The binding energy Ebinding was obtained as the energy of the complex minus the sum of individual Itra and CD energies according to Ebinding = Eguest/CD – (Eisolated guest + Eisoiated CD), RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY
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For higher complexation, β-CD molecules were introduced manually to the already optimized 1 : 1 complex in different possible orientations. The substrate was optimized alone first, and then the resulting complex was optimized free of any restrictions. The No. 7
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EL-BARGHOUTHI et al. lnK* 14
10
–16
12
–8
8
0
16
x
10 –10
8
A
6 4 3.05
*
*
* 3.10
3.15
*
*
–20
3.20 3.25 103/T, K–1
B
–30
Ebinding, kcal/mol Fig. 2. Plot of ln K *11 , ln K *12 , and ln K *13 against 1000/T (K) of the Itra/HP-β-CD complexes in 0.1 M phosphate buffer at pH 7.0.
Fig. 3. The Itra/β-CD binding energy (Ebinding) plotted against the x-coordinate for approaches A and B using an MM+ force field.
Fig. 4. Side views of optimal Itra/β-CD complex configurations obtained using an MM+ force field for 1 : 1, 1 : 2, and 1 : 3 complexes.
binding energy (Ebinding) was plotted against the x-coordinate for approaches A and B, as shown in Fig. 3. The large barriers obtained in Fig. 3 (~30 kcal/mol) indicate that the bi ring of Itra can be included within the cavity neither from the narrow nor from the wide rims. After removing all restrictions on the interacting systems at the energy minima, the binding energies corresponding to optimal configurations for the approaches A and B were –34.9 and –33.6 kcal/mol, respectively. This indicates that approach A yields the most probable 1 : 1 complex geometry. However, the difference in energy between the two approaches is 1.3 kcal/mol only; thus, isomeric configurations are possible. The obtained high value of binding energy may explain the high formation constant of the complex. Figure 4 depicts side views of the optimal 1 : 1, 1 : 2, and 1 : 3 complex configurations obtained for neutral Itra with β-CD. For the 1 : 1 complex, the results indicate that optimal interaction involves the inclusion of the hydrophobic benzene ring (in the methoxy-benzene
group) and part of the piperazine group into the cavity of β-CD. For the 1 : 2 complex, the second β-CD was found to interact with the triazolone ring and its neighboring benzene. The triazole ring seems to interact with the third β-CD. ACKNOWLEDGMENTS The authors wish to thank Hashemite University and Jordanian Pharmaceutical Manufacturing for financial support. REFERENCES 1. D. Duchene, Cyclodextrins and Their Industrial Uses (Editions de Sante, Paris, 1987). 2. J. Szejtli, Chem. Rev. 98, 1743 (1998). 3. K. Uekama, F. Hirayama, and T. Irie, Chem. Rev. 98, 2045 (1998).
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INCLUSION COMPLEXATION OF ITRACONAZOLE 4. I. Tabushi, Y. Kiyousuke, T. Sugimoto, and K. Yamamura, J. Am. Chem. Soc. 100, 916 (1978). 5. M. V. Rekharsky and Y. Inoue, Chem. Rev. 98, 1875 (1998). 6. J. Van Cutsem, Mycoses 32, 14 (1989). 7. J. Peeters, P. Neeskens, J. P. Tollenaere, et al., J. Pharm. Sci. 91, 1414 (2001). 8. K. Miyake, T. Irie, H. Arima, et al., Int. J. Pharm. 179, 237 (1999). 9. N. L. Allinger, Y. H. Yuh, and J. H. Lii, J. Am. Chem. Soc. 111, 8551 (1989).
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10. C. Betzel, W. Saenger, B. E. Hingerty, and G. M. Brown, J. Am. Chem. Soc. 106, 7545 (1984). 11. T. Higuchi and K. A. Connors, Adv. Anal. Chem. Instrum. 4, 117 (1965). 12. A. Yoshida, M. Yamamoto, T. Itoh, et al., Chem. Pharm. Bull. (Tokyo) 38, 176 (1990). 13. F. Kopecky, B. Kopecky, and P. Kaclik, J. Inclusion Phenom. 39, 215 (2001). 14. B. W. Muller and E. Albers, Int. J. Pharm. 79, 273 (1992).
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