Physicochemical investigation of the effects of water-soluble polymers on vinpocetine complexation with β-cyclodextrin and its sulfobutyl ether derivative in solution and solid state

European Journal of Pharmaceutical Sciences 20 (2003) 253–266

Physicochemical investigation of the effects of water-soluble polymers on vinpocetine complexation with ␤-cyclodextrin and its sulfobutyl ether derivative in solution and solid state Laura S.S. Ribeiro a , Domingos C. Ferreira b , Francisco J.B. Veiga a,∗ a

Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra 3000, Portugal b Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal Received 11 November 2002; received in revised form 1 July 2003; accepted 9 July 2003

Abstract The studies reported in this work aimed to elucidate the inclusion complex formation of vinpocetine (VP), a poorly water-soluble base type drug, with ␤-cyclodextrin (␤CD) and its sulfobutyl ether derivative (sulfobutyl ether ␤-cyclodextrin (SBE␤CD)), with or without water-soluble polymers (PVP and HPMC), by thoroughly investigating their interactions in solution and solid state. Phase solubility studies were carried out to evaluate the solubilizing power of both cyclodextrins (CDs), in association with water-soluble polymers, towards VP and to determine the apparent stability constants (Kc ) of the complexes. SBE␤CD showed higher solubilizing efficacy toward VP than the parent ␤CD due to its greater solubility and complexing abilities, what was reflected in higher Kc values. Improvement in Kc values for ternary complexes clearly proves the benefit on the addition of water-soluble polymers to promote higher complexation efficiency. VP–CDs (1:1) binary and ternary systems were prepared by physical mixing, kneading, co-evaporation, and lyophilization methods. In the solid state, drug-carrier interactions were studied by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffractometry (XRD) and Fourier-transform infrared spectroscopy. The results of these analysis suggested the formation of new solid phases, some of them in amorphous state, allowing to the conclusion of strong evidences of binary and ternary inclusion complex formation between VP, CD and water-soluble polymers, particularly for co-evaporated and lyophilized binary and ternary products. © 2003 Elsevier B.V. All rights reserved. Keywords: Vinpocetine; ␤-Cyclodextrin; Sulfobutyl ether ␤-cyclodextrin; Water-soluble polymers; Physicochemical characterization; Binary and ternary complexes

1. Introduction Monomolecular encapsulation by means of monomolecular inclusion complex formation has offered promise for the development of new dosage forms. Generally, the monomolecular inclusion complex involves the spatial entrapment of a single guest molecule in the cavity of one host molecule without any covalent bonds being formed (Uekama and Otagiri, 1987). Cyclodextrins (CDs) are known to form inclusion complexes with a variety of guest molecules both in solution and in the solid state, in which the hydrophobic environment of the CD cavity surrounds each guest molecule. This can lead to alteration of physical, ∗ Corresponding author. Tel.: +351-2398-37850; fax: +351-2398-37731. E-mail address: [email protected] (F.J.B. Veiga).

0928-0987/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0928-0987(03)00199-4

chemical and biological properties of guest molecules, and can eventually have considerable pharmaceutical potential. Thus, the hydrophilic CDs have been extensively used to enhance the oral bioavaibility of several drugs. These improvements are mainly ascribable to the increase in solubility and wettability of the drugs through the formation of inclusion complexes (Hirayama and Uekama, 1999). For a series of reasons (price, availability, approval status and cavity dimensions) the parent ␤-cyclodextrin (␤CD) is the most widely used. However, its anomalous low aqueous solubility (and low solubility of the most complexes) is a serious barrier in its wider utilization (Szejtli, 1994). To overcome those difficulties, chemical modifications have been made to enhance and expand the functionalities of CDs. A significant effort has focused on modifying the solubility of CDs, and thus their complexed guest molecules. One of the most prominent groups of modified CDs, as

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far as pharmaceutical applications are concerned, are the sulfobutyl-substituted CDs, among which is the sulfobutyl ether ␤-cyclodextrin (SBE␤CD). SBE␤CD is a polyanionic highly water-soluble CD derivative, with an average degree of substitution of seven and greater solubility in water than the parent CD (␤CD) (Rao et al., 2001). The inclusion ability of SBE␤CD is generally greater than that of ␤CD due to the hydrophobic butyl side arms that extend the hydrophobic cavity of the CD (Mosher and Thompson, 2002). Unfortunately, the complexation efficiency of CDs is rather low and consequently significant amounts of CDs are needed to solubilize small amounts of water-insoluble compounds. However, enhanced complexation can be obtained by formation of ternary complexes (or co-complexes) between a drug molecule, CD molecule and a third component. For instance, the addition of small amounts of a water-soluble polymer to an aqueous complexation medium, followed by heating the medium in an autoclave, can significantly increase the stability constant of drug–CD complexes (Loftsson and Brewster, 1996). Vinpocetine (VP) is a vincamine derivative used for the treatment of disorders arising from cerebrovascular and cerebral degenerative diseases (Subhan and Hindmarch, 1985). VP is claimed to increase the cerebral flow in the ischaemia affected area of patients with cerebrovascular disease, to decrease platelet aggregability in patients with transient ischaemic attack or stroke, to increase red blow cell deformability in stroke patients, to have neuroprotective abilities and protective effect against brain ischaemia (Feigin et al., 2001). VP is usually available in the market in tablets containing 5 mg of active ingredient. However, existing formulations exhibit poor oral bioavaibility (∼6.7%) (Grandt et al., 1989) and poor absorption, due to its reduced solubility (Kata and Lukács, 1986). To overcome these difficulties, several attempts have been made, namely the formation of VP salts with citrate and phosphate (Calvo and Manresa, 1988) and its complexation with CDs (Kata and Gyorgy, 1982; Kata and Lukács, 1986). The purpose of this work was to examine the potential of ␤CD and its amorphous derivative SBE␤CD, in association with water-soluble polymers (PVP and HPMC) as solubilizing, complexing and amorphizing agents for VP. The water-soluble polymers were added with the aim of increasing the complexation efficiency of CDs toward VP.

2. Materials and methods 2.1. Materials ␤-Cyclodextrin (Kleptose® ; Lot 341001; MW 1135) and sulfobutyl ether ␤-cyclodextrin (CaptisolTM ; Lot CY-03A-119015; TDS 6.8; MW 2160) were kindly supplied by Roquette (Lestrem, France) and Cydex (Kansas City, USA), respectively. Vinpocetine (VP, Lot CV/VP010701;

MW 350.46) was purchased from Covex (Madrid, Spain). Polyvinylpyrrolidone K30 (PVP), hydroxypropylmethylcellulose (HPMC) 4000 cps and tartaric acid (TA) were obtained from Sigma Chemical Co. (St. Louis, USA).

2.2. Phase solubility studies Phase solubility studies in deionized water at room temperature (22 ± 1 ◦ C) were carried out for both binary and ternary systems according to the method of Higuchi and Connors (1965). Excess amounts of VP were weighted into 20 ml glass flasks to which were added 10 ml of aqueous solutions containing increasing amounts of ␤CD (0.001–0.015 or 0.001–0.028 M in the presence of polymers) or SBE␤CD (0.001–0.06 M), with or without a fixed polymer concentration of 0.25% (w/v) PVP and 0.1% (w/v) HPMC. Those polymer concentrations were selected on the basis of preliminary studies carried out between VP and PVP or HPMC, since no further improvement in the solubility values of VP was achieved by increasing polymer concentrations. For binary systems, glass containers were sealed and mechanically stirred until reaching equilibrium (about 72 h). In the case of ternary systems, glass containers were sealed and heated in an autoclave at 120 ◦ C for 20 min and then allowed to equilibrate for 72 h. All suspensions were filtered through a 0.45 ␮m membrane filter (Millipore) and analyzed spectrophotometrically (UV-1603, Shimadzu, Kyoto, Japan) at 316 nm for drug content. Three replicates have been made for each experience. The apparent stability constants (Kc ) were estimated from the straight line of the phase solubility diagrams according to the equation of Higuchi and Connors (1965).

2.3. Preparation of VP–CD binary and ternary solid systems Three different techniques were applied to obtain VP–CD solid complexes, using tartaric acid as an acidifier of the complexation medium. Physical binary and ternary mixtures with and without the addition of tartaric acid were prepared in the same weight ratio of the complexes for further comparison. 2.3.1. Physical binary and ternary mixtures (PM) Physical mixtures (PM) were prepared by homogeneous blending in a mortar of previously sieved (63–120 ␮m sieve granulometric fraction) and weighted VP and SBE␤CD. For ternary PMs, 15% (w/w) of PVP or 6% (w/w) of HPMC were added. PMs containing a VP:TA ratio of 2:1 (w/w), exactly the same amount of tartaric acid as present in all co-evaporated, lyophilized and kneaded (KN) systems described below, were also prepared for further comparison. All mixing procedures were performed adopting the geometric method.

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2.3.2. Kneaded binary and ternary products (KN) Kneaded products were prepared from binary and ternary PM by adding a small volume of 17% (w/v) TA solution corresponding to a 2:1 (w/w) VP:TA ratio. After wetting the PM in a ceramic mortar, the resultant systems were vigorously kneaded for 30 min to produce a homogeneous dispersion. Once homogeneous slurry was obtained, samples where dried at 40 ◦ C for 48 h. 2.3.3. Co-evaporated (COE) binary and ternary products Equimolar amounts of CDs and VP were dissolved in water and in 1.5% (w/v) TA solution, respectively. The resulting mixture was stirred at 100 rpm and a temperature of 60 ◦ C for 3 h and the obtained clear solution was evaporated under vacuum at 50 ◦ C in a rotatory evaporator (Heidolph, Laborota). For ternary products, equimolar amounts of CDs and VP were dissolved in 0.25% (w/v) of PVP solution or 0.1% (w/v) of a HPMC solution and in 1.5% (w/v) TA solution, respectively. The resulting solution was mixed and sonicated for 15 min and then heated in an autoclave (Uniclave 88) at 120 ◦ C for 20 min. After an equilibrium period of 72 h at room temperature, the clear solution was evaporated under vacuum at 50 ◦ C in a rotatory evaporator. All solid residues were further dried at 40 ◦ C for 48 h. 2.3.4. Lyophilized (LPh) binary and ternary products Equimolar amounts of CDs and VP were dissolved in water and in 1.5% (w/v) TA solution, respectively. The two solutions were sonicated for 15 min and mixed for 2 h at 50 ◦ C. Furthermore, the resultant clear solution was frozen by immersion in an ethanol bath (Shell Freezer, Labconco, Freezone® model 79490) at −50 ◦ C and then the frozen solution was lyophilized in a freeze–dryer (Lyph-lock 6 apparatus, Labconco) for 72 h. For ternary products, equimolar amounts of CDs and VP were dissolved in 0.25% (w/v) of PVP solution or 0.1% (w/v) of a HPMC solution and in 1.5% (w/v) TA solution, respectively. The resulting solution was mixed and sonicated for 15 min and then heated in an autoclave at 120 ◦ C for 20 min. After an equilibrium period of 72 h at room temperature, the clear solution was frozen by immersion in an ethanol bath at −50 ◦ C and subsequently lyophilized in a freeze–dryer for 72 h. All resultant dried systems were sieved and fractions smaller than 100 ␮m were collected for further studies. 2.4. Scanning electron microscopy (SEM) The surface morphology of the raw materials and of the binary and ternary systems was examined by means of a scanning electron microscope (Jeol, JSM 5310, Tokyo, Japan). The samples were fixed on a brass stub using double-sided tape and then made electrically conductive by coating in a vacuum with a thin layer of gold. The photographs were taken with a Pentax (model MZ-10) camera at an excita-

255

tion voltage of 10 KV and magnifications factors of 200 and 2000. 2.5. Differential scanning calorimetry (DSC) The DSC curves of pure materials, binary and ternary systems were recorded on a Shimadzu DSC-50 System (Shimadzu, Kyoto, Japan) with a DSC equipped with a computerized data station TA-50WS/PC. The thermal behavior was studied by heating all samples (1 mg of VP or its equivalent) in a sealed aluminum pan, using an empty pan sealed as reference, over the temperature range of 30–250 ◦ C, at a rate of 10 ◦ C/min and under a nitrogen flow of 20 cm3 /min. Indium (99.98%, mp 156.65 ◦ C, Aldrich® , Milwaukee, USA) was used as standard for calibrating the temperature. Results were obtained in triplicate. 2.6. X-ray diffractometry (XRD) Powder X-ray diffraction patterns of VP, CDs, polymers and, binary and ternary systems were analyzed at room temperature with an automated Philips X’Pert (model PW 3040/00) diffractometer system equipped with Co as anode material and a graphite monochromator using a voltage of 40 KV and a current of 35 mA. The diffractograms were recorded in the 2θ angle range between 5 and 50◦ and the process parameters were set as: scan step size of 0.025 (2θ), scan step time of 1.25 s and time of acquisition of 1 h. A software package attached to the diffractometer was used to calculate peak heights of all diffraction patterns. The XRD traces of all raw materials and binary and ternary systems were compared with regard to peak position and relative intensity, peak shifting and the presence and/or lack of peaks in certain regions of 2θ values. Crystallinity was determined by comparing some representative peak heights in the diffraction patterns of the binary and ternary systems with a reference. The relation used for the calculation of the crystallinity was the relative degree of crystallinity (RDC) = ISA /IREF , where ISA is the peak height of the sample under investigation and IREF is the peak height of the same angle for the reference with the highest intensity (Ryan, 1986). To identify the possible interactions between VP, ␤CD, SBE␤CD, HPMC and PVP, VP was used as a reference sample for calculating RDC of all binary and ternary systems. In the case of VP–␤CD binary and ternary systems, ␤CD was also used as a reference for the calculation of RDC values. The crystallinity of PM and of the respective products, concerning to VP, was compared by considering these systems as reference samples for the corresponding binary and ternary products, as described by Veiga et al. (1998). Statistical comparison of the results from multiple products were made by one-way analysis of variance (ANOVA) and the significant level set at P < 0.05, followed by Bonferroni’s multiple comparison test. The statistical analysis were done using a GraphPad Prism® , Version 3.03 software.

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related to the inclusion ability of the CDs molecules in water. The slope values were in all diagrams less than one suggesting the formation of 1:1 stoichiometry complexes in solution and allowing to the calculation of the apparent stability constants (Kc ) of the drug–CD complexes by the Higuchi and Connors equation. The Kc values of VP–CD binary and ternary complexes and VP solubility values in solutions of different composition are presented in Table 1. The binding potential of SBE␤CD was higher than the parent CD because the charged groups of SBE␤CD are appropriately spaced from the cavity and the hydrophobicity around the cavity increases due to the presence of alkyl chains (Uekama et al., 1998). Therefore, Kc values of VP–SBE␤CD binary and ternary complexes were greater than those obtained with VP–␤CD binary and ternary complexes since the complexation with the SBE␤CD involves the CD cavity, as well as the hydrophobic butyl side arms that extend the hydrophobic cavity of the CD. The addition of water-soluble polymers to the CD solution did not change the type of phase-solubility diagrams obtained for binary systems and always resulted in a Kc increase that varied from a minimum of 15% to a maximum

2.7. Fourier-transform infrared spectroscopy (FT-IR) The FT-IR spectra of the samples were recorded on a Jasco FT/IR-420 spectrophotometer equipped with a single reflection horizontal ATR (MiracleTM , PIKE Technologies) applying Fourier-transformation of 16 scans. The scanning was done in 4000–600 cm−1 range at 4 cm−1 resolution.

3. Results and discussion 3.1. Phase solubility studies The phase-solubility studies diagrams obtained with both CDs (␤CD and SBE␤CD) with and without water-soluble polymers (PVP and HPMC) are shown in Fig. 1. They displayed AL type (Higuchi and Connors, 1965) equilibrium phase solubility diagrams for both VP–␤CD and VP–SBE␤CD binary and ternary systems, showing that VP solubility increase linearly as a function of CD concentration and that soluble complexes were formed without occurrence of precipitation in the range of CD concentration used. The increment of VP solubility seems to be

0,16

VP concentration (mM)

VP concentration (mM)

0,6 0,12

0,08

0,04

0,4

0,2

0

0 (A)

0

0,005

0,01

0,015

0,02

0,025

0,03

(B)

β-CD concentration (mM)

0

0,02

0,04

0,06

SBEβ-CD concentration (mM)

Fig. 1. Phase solubility diagrams for vinpocetine (VP) at room temperature in the presence of (A) ␤-cyclodextrin (␤CD) and (B) sulfobutyl ether ␤-cyclodextrin (SBE␤CD) without water-soluble polymers (䉬) and with 0.25% (w/v) PVP (䊏) or 0.1% (w/v) HPMC (䉱). Each point is the mean of three determinations. Table 1 Values of apparent stability constant (Kc ± standard deviation) and VP solubility in VP–CD binary and ternary complexes ␤CD

No polymer 0.25% (w/v) PVP 0.10% (w/v) HPMC a b c d

SBE␤CD

S1 a

S2 b

S1 /S2 c

Kc (M−1 )

KTS /KBS d

S1 a

S2 b

S1 /S2 c

Kc (M−1 )

KTS /KBS d

5.1 8.9 7.2

11.7 22.4 32.3

– 1.09 1.71

75 ± 3 142 ± 4 242 ± 7

– 1.89 3.23

5.1 8.9 7.2

140.8 205.7 162.5

– 1.30 1.10

340 ± 8 490 ± 12 390 ± 8

– 1.44 1.15

VP solubility (␮g/ml) in water or in aqueous polymer solutions. VP solubility (␮g/ml) in CD solutions (15 mM ␤CD or 60 mM SBE␤CD) with and without polymers. S1 /S2 is the ratio of VP solubility achieved in VP–CD ternary systems and the corresponding sum of values in CD and polymer solutions. KTS /KBS is the ratio of Kc for VP–CD ternary and binary complexes.

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of 323%, depending on the CD and water-soluble polymer considered. The observed enhancement of Kc upon addition of the polymers shows that they are able to interact in a different way with drug–CD binary complexes depending on their structures, since the polymers can establish different interactions with CD and drug molecules such as hydrophobic bonds, Van der Walls dispersion forces or hydrogen bonds (Faucci and Mura, 2001). The different complexation efficiency of PVP and HPMC in the presence of ␤CD and SBE␤CD may be due to structural and polarity differences of the two CD molecules that could be in the origin of different type of linkings established with the polymers and the drug. Therefore, the solubilizing effect of CDs was increased in the presence of both 0.25% (w/v) PVP and 0.1% (w/v) HPMC. Consequently, a synergistic effect on VP solubility was observed in the presence of CDs and water-soluble polymers since the solubility values achieved in the presence of both CDs and polymers were higher than the sum of the contribution solubility values obtained with the CD and polymer solutions (Loftsson et al., 1999). The improvement on VP solubility in the presence of both CD and polymer systems varied between 9 and 71%, in comparison to the sum of VP solubilities obtained in the CD and polymer solutions. Moreover, the presence of polymers in the complexation medium changed the ␤CD water solubility making possible an increase on VP solubility under a wider range of ␤CD concentration (0–28 mM), reaching solubility values of 30.7 and 47.5 ␮g/ml in a 28 mM ␤CD solution with 0.25% (w/v) PVP and 0.1% (w/v) HPMC, respectively. 3.2. Scanning electron microscopy (SEM) Due to the existence of strong morphological similarities between binary and ternary products, the SEM microphotographs of the VP–CD binary products have been selected and are represented in Fig. 2. From SEM analysis, it can be seen that pure VP particles appeared as small crystals (1–30 ␮m) of irregular shape, ␤CD particles consisted of three-dimensional parallelogram crystals of irregular size (20–250 ␮m), whereas hollow spherical particles with a large size distribution (1–180 ␮m) were evident in the SBE␤CD microphotographs. Microscopic examination of VP–CD PMs showed the presence of VP crystals mixed and adhered on the surface of CD particles, revealing no apparent interaction between both species in the solid state. On the contrary, a drastic change in the original morphology and shape of both VP and CD particles were observed in all binary and ternary products, being the morphology influenced by the preparation method adopted. In the KN binary products, it was still possible to distinguish VP crystals as agglomerates on the surface of CD particles that had lost their original shapes. The microphotographs of binary and ternary COE products revealed the formation of large masses of undifferentiated particles, which were different from those of raw materials. Finally, the lyophilization technique gave rise to amorphous pieces of irregular and small

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size with a lamellate aspect common for binary and ternary products. In both COE and LPh binary and ternary products, it could just be distinguished a single component. The drastic change of the particle shape and aspect in VP–CD COE and LPh products, indicative of the presence of a new solid phase, could be simply a consequence of a crystalline habitus change in those systems or it may support the evidence of the existence of a single phase (Fernandes et al., 2002), therefore we proceed to complementary solid-state characterization studies. 3.3. Differential scanning calorimetry (DSC) The DSC profiles of pure components and of the respective binary and ternary systems in the melting range of the drug and dehydration of the carrier are shown in Figs. 3 and 4. The thermal curve of pure VP was typical of a crystalline anhydrous substance with a sharp endothermic peak at 149.3 ± 0.6 ◦ C corresponding to the melting point of the drug. The DSC curve of ␤CD showed the liberation of crystal water as an endothermal effect peaked at about 135 ◦ C, whereas broader endotherms were associated with water loss from amorphous SBE␤CD, PVP and HPMC. The comparison of DSC curves from binary systems with those belonging to ternary systems did not result in significant differences. Both characteristics peaks of VP (drug melting) and CDs (water loss) were clearly distinguishable in binary and ternary PMs, being the DSC curves of those the superposition of the individual components. Concerning the binary and ternary PM with addition of TA, the thermal characteristic peak of VP was shifted to lower temperatures around 146.0–148.6 ◦ C and its intensity was reduced. These little changes relative to the peak of pure VP may suggest a weak interaction between the components of the PM with TA by mechanical activation during the mixing or heating for DSC scanning (Dollo et al., 1999; Mura et al., 2001). As far as the kneading method is concerned, a small broad peak was still obvious in the dehydration peak of SBE␤CD in the DSC curves of both binary and ternary KN products. Despite of the lower size and shifts to lower temperatures (around 144.2–148.9 ◦ C), the small peak correspondent to the melting of free drug suggests that, as for PMs, there is no inclusion compound formed in either system even though drug–CD interaction is expressive. The complete disappearance of the drug endothermal effect was nevertheless observed in VP–␤CD binary and ternary KN products. However, having in account the results of SEM microphotographs, where the presence of VP were still detectable, we can assume to have occurred a reduction of drug crystallinity or probably to have occurred a partial dispersion at a molecular level in the solid product (Ahmed et al., 1998). DSC curves of VP–CD COE and LPh products exhibited as well the complete disappearance of the endothermic melting peak of VP and the only difference between them was in respect to the dehydration phenomenon of the products, which is indicative of a different degree of water

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Fig. 2. Scanning electron micrographs of VP–␤CD binary systems: (a) physical mixture, (b) kneaded, (c) co-evaporated and (d) lyophilized products; and VP–SBE␤CD binary systems: (e) physical mixture, (f) kneaded, (g) co-evaporated and (h) lyophilized products.

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259

Fig. 3. Differential scanning calorimetry curves of vinpocetine (VP), ␤-cyclodextrin (␤CD), water-soluble polymers (PVP and HPMC), and VP–␤CD binary and ternary systems: physical mixtures (PM), physical mixtures with tartaric acid (PM + ac), kneaded (KN), co-evaporated (COE) and lyophilised (LPh) products.

Fig. 4. Differential scanning calorimetry curves of vinpocetine (VP), sulfobutylether-␤-cyclodextrin (SBE␤CD), water-soluble polymers (PVP and HPMC), and VP–SBE␤CD binary and ternary systems: physical mixtures (PM), physical mixtures with tartaric acid (PM + ac), kneaded (KN), co-evaporated (COE) and lyophilised (LPh) products.

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elimination by the preparation method. As the disappearance of an endothermic peak may be attributed to an amorphous state and/or to an inclusion complexation (Kurazumi et al., 1975), these results suggest that only VP–CD COE and LPh products can be considered as true inclusion complexes, differing from simple PMs. 3.4. X-ray diffractometry (XRD) The X-ray diffraction patterns of VP, ␤CD, SBE␤CD, PVP, HPMC and binary and ternary systems are shown in Figs. 5 and 6 and the RDC values and peak intensities of VP–CD systems are presented in Tables 2 and 3. The X-ray diffraction pattern of VP revealed several high intensity reflections indicative of its crystalline character whose intensities and peak positions are summarized in Table 3. Also ␤CD has shown a crystalline diffractogram with the most

relevant diffraction peaks at 7.313, 10.387, 12.387, 17.913, 14.587, 21.838 and 22.838◦ (2θ), while a halo-pattern was recorded for SBE␤CD, PVP and HPMC demonstrating their amorphous states. Comparing the diffraction patterns of pure components with those of VP–␤CD binary and ternary systems, we deduced that there were no marked dissimilarities between the diffraction patterns of binary and ternary PMs, being all of them the superposition of the pure components. However, a lower intensity of their diffraction peaks and overlapping of some VP peaks with the peaks of ␤CD was also observed, what was attributed to the reduction in particle size during the preparation of the PMs and to the dilution of the pure crystalline components. The RDC values of all PMs were statistically equivalent (P > 0.05) either considering VP or ␤CD as references. The diffraction patterns of KN and COE binary products displayed a crystalline state

Fig. 5. Powder X-ray diffraction patterns of VP–␤CD binary systems (A), VP–␤CD–PVP ternary systems (B) and VP–␤CD–HPMC ternary systems (C): physical mixtures (PM), physical mixtures with tartaric acid (PM + ac), kneaded (KN), co-evaporated (COE) and lyophilised (LPh) products (arrows indicate the position of the missing diffraction peaks).

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261

Fig. 6. Powder X-ray diffraction patterns of VP–SBE␤CD binary systems (A), VP–SBE␤CD–PVP ternary systems (B) and VP–SBE␤CD–HPMC ternary systems (C): physical mixtures (PM), physical mixtures with tartaric acid (PM + ac), kneaded (KN), co-evaporated (COE) and lyophilised (LPh) products (arrows indicate the position of new diffraction peaks).

but in comparison with the diffractogram of the correspondent PM it was possible to observe the disappearance of some diffraction peaks of ␤CD and VP at 7.313, 16.138 and 27.638◦ (2θ), signalized in Fig. 5 with arrows. Moreover, as

stated by the peak intensities values of Table 3, almost all diffraction peaks were significantly reduced, what was reflected by lower RDC values than the VP–␤CD binary PM (P < 0.001). The diffraction pattern of VP–␤CD ternary

Table 2 Relative degree of crystallinity (RDC) for VP–CD binary and ternary systems Reference used

␤CD VP PM

VP–␤CD

VP–␤CD–PVP KNb

COEc

LPhd

PMa

KNb

COEc

LPhd

PMa

KNb

COEc

LPhd

1.089 0.543 –

0.687 0.328 0.604

0.672 0.313 0.577

0.529 0.303 0.557

1.097 0.568 –

0.625 0.340 0.599

0.551 0.291 0.513

0.502 0.265 0.466

1.081 0.518 –

0.669 0.335 0.646

0.596 0.298 0.574

0.499 0.202 0.389

0.178 0.741

0.172 0.712

VP–SBE␤CD VP PM a b c d

VP–␤CD–HPMC

PMa

0.240 – Physical mixtures. Kneaded products. Co-evaporated products. Lyophilized products.

0.204 0.851

VP–SBE␤CD–PVP 0.195 0.815

0.170 0.711

0.238 –

0.193 0.810

VP–SBE␤CD–HPMC 0.188 0.792

0.159 0.670

0.241 –

0.194 0.804

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Table 3 Peak intensities of vinpocetine (VP) in the X-ray diffraction patterns for VP–CD binary and ternary systems Peak position, 2θ (◦ )

14.387 20.438 21.862 27.638

VP

3821 2826 5264 3578

VP–␤CD

VP–␤CD–PVP KNb

COEc

LPhd

PMa

KNb

COEc

LPhd

PMa

KNb

COEc

LPhd

3578 1822 2861 1434

2773 1202 1650 702

2529 1556 1729 732

1001 1428 1594 689

3581 1807 2989 1405

2228 1357 1534 804

1608

816 1488 1064 678

3575 1837 2732 1463

2499 1311 1567 783

1977 1537 1764 884

816 1488 1064 678

VP–SBE␤CD 14.387 16.138 17.313 20.438 21.862 27.638 a b c d

3821 4236 2179 2826 5264 3578

VP–␤CD–HPMC

PMa

836 917 660 964 1261 836

556 566 516 800 1073 619

– 1791 953

VP–SBE␤CD–PVP 540 557 527 855 1028 610

531 470 475 784 896 558

844 958 661 954 1253 813

633 621 621 865 1015 607

VP–SBE␤CD–HPMC 590 543 542 834 992 637

513 515 513 730 839 569

827 875 658 974 1268 859

587 609 578 866 1020 606

565 547 513 815 939 608

547 533 510 795 903 573

Physical mixtures. Kneaded products. Co-evaporated products. Lyophilized products.

KN and COE products showed the position maintenance of the main diffraction peaks when confronted with the correspondent binary ones, though some characteristic peaks of VP were also absent. A marked decrease in the crystalline character was also observed for COE products, suggesting a disorder phenomenon upon complexation (Cortés et al., 2001). Furthermore, for binary and ternary VP–␤CD LPh products the obtained patterns were diffused indicating the amorphous state reached by the lyophilization technique. This amorphousness was well reflected by lower RDC values, even more significant for VP–CD ternary LPh product (P < 0.001). In the diffractogram of VP–SBE␤CD PMs, the presence of free crystalline drug was revealed by few and broad peaks of low intensity that emerged on the diffuse background of the amorphous carriers indicating a clear loss of VP crystallinity. In fact, the X-ray diffraction patterns of the PMs contain the principal diffraction peaks of VP and were apparently only the superposition of each component, with a marked decrease in their intensities. From the RDC values of the PMs, it was observed that the degree of crystallinity of both binary and ternary PMs were similar (P > 0.05), as can be confirmed by the peak height of the principal diffraction peaks of VP. The diffractograms of VP–SBE␤CD KN products exhibited lower RDC values than the respective PMs (P < 0.05), without significant differences between the crystallinity of KN binary and ternary products (P > 0.05). The reduction of RDC values from KN products in comparison with the corresponding PMs could be explained by the presence of reciprocal interactions in the solid state between host and guest, namely the formation of mixed particles during the drying process, as observed in SEM analysis (Veiga et al., 1998). Complete VP amorphization was also detected in the VP–SBE␤CD and VP–SBE␤CD–PVP COE products and in all VP–SBE␤CD LPh products, showing a typical flat

behavior that confirms the strong ability of the amorphous carrier SBE␤CD to induce drug amorphization (Rao et al., 2001), as a result of the preparation technique. As the amorphization of the drug can be a consequence of the lyophilization process, the X-ray data cannot discriminate whether the VP–CDs LPh products obtained are true inclusion complexes or homogeneous dispersed mixtures of the amorphous components (Redenti et al., 1996). Nevertheless, having in account the results of SEM and DSC analysis, as well as the lack of some important diffraction peaks in VP–␤CD binary and ternary COE products, and the complete drug amorphization in the majority of VP–SBE␤CD COE, we can assume the formation of some new solid phases that may be credited to the formation of inclusion complexes, in which VP is at least partially entrapped in the CD cavity (Charoenchaitrakool et al., 2002). On the other hand, the diffractogram of VP–SBE␤CD–HPMC COE product showed the appearance of new diffraction peaks at 23.912, 26.487, 29.987 and 43.238 (2θ) distinct from those that emerged on the diffraction patterns of the respective PM (arrows in Fig. 6). Such results demonstrated that the VP–SBE␤CD–HPMC COE product is a new chemical entity, different from the original substances (Lu et al., 2000) and may be considered an indirect proof of complexation between VP, SBE␤CD and HPMC. In what concerns the crystalline character of the systems, VP–␤CD binary and ternary systems are markedly more crystalline than VP–SBE␤CD binary and ternary ones (P < 0.001). This effect is clearly stated by higher RDC values for the first systems. In a general way, the RDC values of COE and LPh binary and ternary products were lower than those of KN products and the respective PMs. The diffraction patterns of LPh ternary systems were superimposable with the corresponding binary ones. In the case of COE products, lower RDC values were obtained for VP–SBE␤CD COE ternary products in comparison with binary ones, because

L.S.S. Ribeiro et al. / European Journal of Pharmaceutical Sciences 20 (2003) 253–266

the RDC values are calculated from the height of the most representative diffraction peak of the drug, but this difference has not a statistical significance (P > 0.05). The amorphous state of the several VP–CDs systems was significantly unlike from each other, being the sequence of increment resumed as follow: VP–␤CD binary and ternary PMs  VP–␤CD binary and ternary KN products < VP–␤CD binary and ternary COE products < VP–␤CD binary LPh product < VP–␤CD ternary LPh products ≤ VP–␤CD binary and ternary PMs < VP–SBE␤CD binary and ternary KN products ≤ VP–SBE␤CD binary and ternary COE products ≤ VP–SBE␤CD binary and LPh products. The extent of the amorphousness was found to be dependent on the selected preparation method and type of CD used.

are very subtle requiring careful interpretation of the spectrum (Hedges, 1998). Moreover, its application is limited for guests that have some characteristic absorption bands such as carbonyl groups (Uekama and Otagiri, 1987). In this study, due to the fact that VP has an ester carbonyl stretching band at 1715 cm−1 , FT-IR could be used to detect guest interactions. The carbonyl-stretching region of IR spectra of VP and its different systems with CDs are presented in Figs. 7 and 8. No significant changes of the characteristic carbonyl-stretching band of VP were observed in any PM either with ␤CD or SBE␤CD indicating that no important interactions should be involved in those products. The spectra of all VP–CD binary and ternary products did no show new peaks indicating that no chemical bonds were created in the formed compounds. Though, IR C=O stretching band was instead highly diminished, broader and shifted to lower frequencies in all spectral patterns of KN, COE and LPh binary and ternary products. It was verified that the magnitude of the alteration of the original C=O stretching band was clearly affected by the preparation method selected. Thus, the decrease in

3.5. Fourier-transform infrared spectroscopy (FT-IR) FT-IR spectroscopy has also been used to assess the interaction between CD and guest molecules in the solid state, since upon complexation of the guest shifts or changes in the absorption spectrum occur. However, some of the changes

VP-βCD

263

VP-βCD-PVP

VP-βCD-HPMC

VP

PVP HPMC βCD PM

PM

PM

PM+ac

PM+ac

KN

KN

COE

COE

PM+ac KN COE LPh

LPh

1800 1600

1400 1200 1000

cm-1

LPh

1800 1600

1400 1200 1000

cm-1

1800 1600

1400 1200 1000

cm-1

Fig. 7. Fourier-transform infrared absorption bands in the 1900–1000 cm−1 region: vinpocetine (VP), ␤-cyclodextrin (␤CD), water-soluble polymers (PVP and HPMC), and VP–␤CD binary and ternary systems: physical mixtures (PM), physical mixtures with tartaric acid (PM + ac), kneaded (KN), co-evaporated (COE) and lyophilised (LPh) products.

264

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Fig. 8. Fourier-transform infrared absorption bands in the 1900–1000 cm−1 region: vinpocetine (VP), sulfobutyl ether ␤-cyclodextrin (SBE␤CD), water-soluble polymers (PVP and HPMC), and VP–SBE␤CD binary and ternary systems: physical mixtures (PM), physical mixtures with tartaric acid (PM + ac), kneaded (KN), co-evaporated (COE) and lyophilised (LPh) products.

the intensity of C=O stretching band and the amplitude of its shift was in the following range KN < COE < LPh, as can be confirmed by the values of C=O stretching band presented in Table 4. Additionally, significant shifts were also observed for the ether C–O–C stretching band ranging from 1020 cm−1 in pure VP to 1035 cm−1 in VP–SBE␤CD

ternary LPh products. Those shifts were in the same magnitude of the ones observed for C=O stretching band. These results served to confirm the existence of strong interactions between VP, CDs and water-soluble polymers, since the spectral changes can be explained by the dissociation of the intermolecular hydrogen bonds of VP through

Table 4 FT-IR characteristics bands (cm−1 ) for vinpocetine (VP) and VP–CD binary and ternary systems IR band

C=O C–O–C

VP

1715 1020

VP–␤CD

VP–␤CD–PVP KNb

COEc

LPhd

PMa

KNb

COEc

LPhd

PMa

KNb

COEc

LPhd

1716 1021

1720 1024

1721 1024

1721 1025

1716 1021

1719 1025

1720 1025

1722 1027

1716 1021

1719 1024

1722 1024

1723 1025

VP–SBE␤CD C=O C–O–C a b c d

1715 1020

VP–␤CD–HPMC

PMa

1716 1022

Physical mixtures. Kneaded products. Co-evaporated products. Lyophilized products.

1718 1028

VP–SBE␤CD–PVP 1724 1031

1724 1032

1716 1022

1719 1031

VP–SBE␤CD–HPMC 1724 1034

1724 1035

1716 1022

1719 1034

1724 1034

1724 1035

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inclusion complexation (Erden and Celebi, 1988) and also may be indicative of VP dispersion as a consequence of the interaction with CDs and polymers, which could result in VP inclusion into the hydrophobic cavity of the carrier (Mura et al., 1998).

265

acknowledge Cydex L.C. (Kansas City, USA) and Roquette (Lestrem, France) for their support providing SBE␤CD and ␤CD, respectively. References

4. Conclusions On the basis of the physicochemical characterization techniques described in this work, the complex formation between VP, CDs (␤CD and SBE␤CD) and water-soluble polymers (PVP and HPMC) was confirmed. Both the stoichiometry and equilibrium constants for the inclusion complexes were evaluated by the phase solubility method. SBE␤CD proved to have better solubilizing and complexing properties for VP than the parent ␤CD, as could be stated by the higher Kc values obtained for both binary and ternary complexes. The greater Kc values found for ternary complexes in comparison with the corresponding binary ones suggest a significant improvement on the complexation efficiency between VP and both CDs by addition of small amounts of water-soluble polymers. The use of SEM, DSC, XRD and FT-IR enabled us to thoroughly elucidate the solid-state interactions of VP–CD binary and ternary systems and suggested the formation of new solid phases, some of them in amorphous state, allowing to the conclusion of strong evidences of binary and ternary inclusion complex formation between VP, CDs and water-soluble polymers, particularly for COE and LPh binary and ternary products. It was verified that apart from the preparation method adopted, the most important factor to differentiate the solid products obtained was the type of CD employed. In fact, VP–SBE␤CD binary and ternary systems exhibited better amorphizing properties for VP than the corresponding solid systems with the parent ␤CD. Taking in account these results, we believe that the interaction of VP with CDs and water-soluble polymers, through the formation of inclusion complexes, can lead to important modifications on the physicochemical and biological properties of the guest molecule, such as the improvement on VP bioavaibility, which might eventually have relevant pharmaceutical potential.

Acknowledgements This work was financially supported by a grant (Praxis XXI/BD/21455/99) from FCT (Fundação para a Ciˆencia e a Tecnologia, Portugal). The authors would like to thank the technical assistance of Dra. Ana Paula Piedade and Dr. Albano from the Instituto Pedro Nunes (Coimbra, Portugal) in recording the X-ray diffractograms and in SEM observations and Dr. Alcino Lopes Leitão (Departamento de Qu´ımica Farmacˆeutica, Faculdade de Farmácia, Universidade de Coimbra) for the kind availability to record FT-IR spectra. We also

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