Interaction of clofibrate with cyclodextrin in solution: phase solubility, 1H NMR and molecular modelling studies

European Journal of Pharmaceutical Sciences, 5 (1997) 215–221

Interaction of clofibrate with cyclodextrin in solution: phase solubility, 1 H NMR and molecular modelling studies ´ S. Anguiano-Igea*, F.J. Otero-Espinar, J.L. Vila-Jato, J. Blanco-Mendez ´ Farmaceutica ´ Departamento de Farmacia y Tecnologıa , Facultad de Farmacia, Universidad de Santiago de Compostela, Campus Universitario, 15706 Santiago de Compostela Spain Received 7 June 1996; accepted 22 January 1997

Abstract Phase solubility diagrams for mixtures of clofibrate and hydroxypropyl-ß-cyclodextrin, dimethyl-ß-cyclodextrin or ß-cyclodextrin in aqueous solution indicated Higuchi A L type behaviour for solutions containing the ß-cyclodextrin derivatives, and B S type behaviour for those containing ß-cyclodextrin, which formed an isolable solid inclusion complex with clofibrate with an apparent stability constant Ks 51315 M 21 . 1 H NMR studies of the latter complex indicated that it has 1:1 stoichiometry, and that host–guest interactions are of a predominantly hydrophobic nature and principally occur between the clofibrate phenyl ring and the interior of the ß-cyclodextrin cavity. The NMR results were used to generate a three-dimensional molecular model of the inclusion complex which has the phenyl ring in the cyclodextrin cavity and the side chain protruding from the wider end of the ß-cyclodextrin molecule  1997 Elsevier Science B.V. Keywords: Clofibrate; Cyclodextrin; Inclusion complexation; 1 H NMR; Molecular modeling; Solubility

1. Introduction In the last few years there has been extensive pharmaceutical research into cyclodextrin inclusion complexes, as evidenced by the appearance during this period of numerous publications and patents describing such complexes of a wide variety of drugs. The benefits that this mode of entrapment can confer on certain molecules are well known, and include increased solubility and bioavailability (Vila-Jato et al., 1986, 1987, 1988; Blanco et al., 1991; TorresLabandeira et al., 1991), attenuated side effects (Szejtli and Szente, 1981; Otero-Espinar et al., 1991) and increased stability (Szejtli et al., 1980; Uekama and Otagiri, 1987). The preparation of solid cyclodextrin inclusion complexes of liquid species is an attractive idea

*Corresponding author. Tel: 134-81-594627. Fax: 134-81-547148. 0928-0987 / 97 / $32.00  1997 Elsevier Science B.V. All rights reserved PII S0928-0987( 97 )00277-7

because it may facilitate drug formulation of solid dosage forms. This approach has already been employed for stabilized volatile substances (Uekama et ¨ al., 1983a,b; Gal-Fuzy et al., 1984; Vikmon et al., 1986). Clofibrate (ethyl 2-(4-chlorophenoxy)-2-methylpropionate) is a bitter-tasting oily liquid with poor water solubility. It is used in treatments for some forms of hyperlipidaemia, for which it is usually formulated in soft gelatin capsules for oral administration. For some pharmaceutical laboratories, investment in the high technology apparatus required for filling of these capsules may not be economically justifiable. Moreover, throughput volume for production of capsules is smaller than for production of other, more classical formulations. Thus, in order to facilitate preparation of traditional formulations of clofibrate, it is of interest to obtain a solid form of this drug through formation of its cyclodextrin inclusion complex. In addition, other desirable drug

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properties may also be enhanced by this mode of entrapment. Before selecting a cyclodextrin for entrapment of a drug molecule, an appreciation of the way in which drug–cyclodextrin interactions occur upon complexation, and how these interactions affect the stability of the inclusion complex, is necessary. Drug–cyclodextrin interactions may be studied by a variety of techniques, most of which examine the behaviour of mixtures of the cyclodextrin (host) and drug species (guest) in solution. One such technique in frequent use is determination of the phase solubility diagram, which can be used to obtain an approximate stability constant for the complex. More direct evidence of complexation can be obtained from proton nuclear magnetic resonance ( 1 H NMR) spectra of the host– guest mixture: this technique allows determination of the orientation of the guest molecule in the cyclodextrin cavity, the stoichiometry of the complex, and a stability constant. Finally, in recent years widespread use has been made of computer aided molecular modelling to rapidly and simply obtain a threedimensional image of the most likely structure of the inclusion complex. A phase solubility diagram technique was employed to estimate the stability constants of clofibrate, ß-cyclodextrin, dimethyl-ß-cyclodextrin and hydroxypropyl-ß-cyclodextrin complexes. The mode of inclusion of clofibrate in the ß-cyclodextrin was studied by 1 H-NMR and molecular modelling.

ing to the method of Higuchi and Connors (1965). Excess amounts of clofibrate were added to a series of solutions containing increasing concentrations of the cyclodextrin and were shaken at 2560.58C. After equilibrium was attained (approx. 7 days) an aliquot was centrifuged for 1 h at 82 555 g (Centrikon (Zurich, Switzerland) T-1075 ultracentrifuge) and 1 ml aliquots of the supernatant were taken and suitably diluted before the concentration of clofibrate in each sample was determined spectrophotometrically ( lmax 5225 nm). Final values of clofibrate solubility are the means of three replicate determinations. The phase solubility diagrams were obtained by plotting the mean solubilities obtained above against cyclodextrin concentration (linear regions were fitted by least-squares regression). The stoichiometry and apparent stability constant of the complex were calculated from the slope and intercept of the straight portion of the phase solubility diagram.

2.3. 1 H NMR studies 1

H NMR spectra of 0.7:1, 1:1, 1.5:1 and 2:1 mole ratios of clofibrate–ß-cyclodextrin in D 2 O were recorded in a Bruker WM 250 (Karlsruhe, Germany) spectrometer at 250 MHz. The chemical shift at 4.8 ppm due to residual solvents (H 2 O and HDO) was used as internal reference (60.002). The signals due to clofibrate and ß-cyclodextrin were respectively assigned by reference to the work of Hassan and Elazzouny (1982); Ueda and Nagai (1980).

2. Experimental procedures

2.1. Materials

2.4. Molecular modelling

Clofibrate was a generous gift from ICI Farma ˜ (Pontevedra, Spain); ß cyclodextrin and Espana heptakis (2,6-di-o-methyl)-ß-cyclodextrin were purchased from Cyclolab (Budapest, Hungary), and hydroxypropyl-ß-cyclodextrin (D.S. 2.7) from Celdex (Tokyo, Japan). Solubility studies used deionized 1 water, and H NMR spectra were of solutions in deuterium oxide (D 2 O, deuterium content 99.8%) from Fluka (Barcelona, Spain).

Molecular modelling was performed using the program ALCHEMY II (Tripos Associates, San Louis, MI, USA), which is a simplified version of the program SIBYL. The geometry of the clofibrate molecule was generated by an energy-minimization subroutine using phenol as the base molecule and adding on the remaining atoms (maximum no. iterations 500; minimizer cut-off 0.01). The molecular geometry of ß-cyclodextrin was obtained by inputting X-ray diffraction data for a monocrystal (Harata et al., 1985). The geometry of the complex is based on the 1 H NMR results and was generated using a manual docking subroutine of ALCHEMY.

2.2. Phase solubility studies Solubility measurements were carried out accord-

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Fig. 1. Phase solubility diagrams for clofibrate with different cyclodextrins at 258C.

3. Results and discussion

3.1. Solubility studies From the solubility curves obtained for the three cyclodextrin hosts (Fig. 1), it was possible to obtain evidence for inclusion-complex formation and to determine which cyclodextrin host was interacting most favourably with the clofibrate guest, and so forming the most stable complex. For ß-cyclodextrin a B S type diagram was obtained (Higuchi and Connors, 1965), while for the two ß-cyclodextrin derivatives, A L type curves were obtained that are typical of formation of soluble inclusion complexes. Apparent stability constants (Ks ) for complexes with each cyclodextrin were calculated from the ascending segments of these solubility curves by assuming that all the complexes had 1:1 stoichiometry; these values are listed in Table 1. The highest value of Ks (Ks 52108) was found for

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dimethyl-ß-cyclodextrin, indicating that clofibrate interacts more strongly with this cyclodextrin. This is probably due to the methylated cyclodextrin’s having ˚ a deeper cavity than ß-cyclodextrin (11 against 8 A) (Harata, 1990), and also its greater hydrophobicity (Smolkova-Keulemansova et al., 1990), which would strengthen hydrophobic interactions between the dimethyl-ß-cyclodextrin and the clofibrate molecule. The lowest value of Ks (Ks 5815) was obtained for the soluble complex of clofibrate and hydroxypropylß-cyclodextrin. We have also obtained a lower value of Ks for hydroxypropyl-b-cyclodextrin than for bcyclodextrin in the case of naproxen (Blanco et al., 1991) although the opposite results have been found by other authors even for naproxen (Bettinetti et al., 1990). Differences in the degree of purity as well as in the substitution degree of hydroxypropyl-b-cyclodextrin may explain these discrepancies. The value of Ks for the clofibrate–ß-cyclodextrin inclusion complex, lies between the values for the latter complexes, and closely agrees with previously reported values (Uekama et al., 1983b; Ben-Amor, 1990). The apparent stability of this complex is sufficiently high for the purposes of this study, and its poorer solubility facilitates isolation of the solid form. Therefore, the inclusion complex of clofibrate with ß-cyclodextrin, a naturally occurring cyclodextrin that is also widely available, was selected for further study with a view to preparation of a solid dosage form of clofibrate. Evaluation of the stoichiometry of the complex from the plateau region of the solubility diagram gave a value of 0.71:1 (clofibrate–ß-cyclodextrin), justifying the assumption of 1:1 stoichiometry for calculation of Ks .

3.2. 1 H NMR spectroscopy studies From the 1 H NMR spectra it is possible to determine the orientation and penetration of the guest molecule in the cavity of the cyclodextrin, and the likely interactions responsible for keeping it there.

Table 1 Summary of the findings from the phase solubility studies Cyclodextrin

KS (M 21 )

S0 (mM)

Smax (mM)

Slope

r

ß-cyclodextrin Dimethyl-ß-cyclodextrin Hydroxypropyl-ß-cyclodextrin

1315 2108 815

0.2930 0.2930 0.2930

0.54 – –

0.2780 0.3820 0.1940

0.9860 0.9920 0.9890

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Plots derived from the NMR spectra (Fig. 2) show how the chemical shifts of the cyclodextrin protons vary with clofibrate concentration: as this concentration increases, the signal due to H 5 , which is initially overlapped by the H 6 signal at 3.82–3.88 ppm, moves to higher field and becomes visible (similar behaviour has been observed previously by Ueda and Nagai (1980); Cabral-Marques et al. (1990). Significant changes are also observed in the signal due to H 3 , whereas H 1 , H 2 and H 4 (located outside the cavity) were relatively unaffected. These observations are in keeping with an inclusion complex which has the phenyl ring of the clofibrate

included in the cavity of ß-cyclodextrin and the cyclodextrin protons would be affected by anisotropic shielding due to the phenyl moiety. For the range of samples studied, the magnitudes of the changes in chemical shift (Dd ) observed for H 3 and H 5 are similar and suggest that the molecule is included deeply into the cyclodextrin cavity. Since the signal due to H 6 , which is located at the narrow end of the molecule, is not significantly shielded by the guest molecule, it is likely that the clofibrate molecule enters from the wider end of the cyclodextrin where the secondary hydroxyls are located. The shifts of all the signals in ß-cyclodextrin to

Fig. 2. Variation of the chemical shift of ß-cyclodextrin protons H 1 H 2 H 3 H 4 H 5 H 6 with clofibrate–ß-cyclodextrin mole ratio (clofibrate / ß-cyclodextrin).

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higher field suggested that a hydrophobic interaction was predominant between the drug and the ß-cyclodextrin (Ueda and Nagai, 1980; Cabral-Marques et al., 1990). In Fig. 3, the effects of the ß-cyclodextrin on the 1 H NMR spectrum of clofibrate are presented for selected signals. Unfortunately, the poor solubility of the clofibrate in D 2 O meant that some proton signals were too weak to be quantitatively analysed under the present experimental conditions. As ß-cyclodextrin concentration increases, the protons H a and H b of the phenyl ring move downfield, which is in keeping with there being predominantly hydrophobic interactions between the clofibrate and the ß-cyclodextrin, such as van der Waals’ forces (Suzuki and Sasaki, ´ 1979; Zhang et al., 1990; Ganza-Gonzalez et al., 1994). The methylene (CH 2 ) of the ethyl group of the side chain is also slightly deshielded, possibly due to steric effects induced by the secondary hydroxyls of the rigid cyclodextrin structure, to which it will be

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very close in the inclusion complex (Otagiri et al., 1975; Uekama et al., 1982). The signals due to the remaining clofibrate protons are barely affected by the complexation. The large Dd observed for the aromatic protons confirm that the host–guest interactions principally involve the phenyl ring. Furthermore, it is clear from Fig. 3 that the largest Dd generally occur for a molar ratio of 1:1, which indicates that a 1:1 complex is formed.

3.3. Molecular modelling Selected views of the three-dimensional structure of the inclusion complex generated from the 1 H NMR results are shown in Figs. 4 and 5. The clofibrate phenyl ring is located deep within the cavity where it can interact with the cyclodextrin groups which line it, while the clofibrate side-chain, whose NMR signals generally showed smaller

Fig. 3. Variation of the chemical shift of clofibrate protons with ß-cyclodextrin–clofibrate mole ratio (ß-cyclodextrin / clofibrate).

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Fig. 4. Molecular model of the clofibrate–ß-cyclodextrin inclusion complex: frontal and lateral views.

Acknowledgments ˜ for The authors are grateful to ICI Farma Espana supplying clofibrate. This work was supported by a grant from Xunta de Galicia (XUGA 20305A91)

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Fig. 5. Molecular model of the clofibrate–ß-cyclodextrin inclusion complex: cross-sectional view.

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