Complexation of Zolpidem with 2‐Hydroxypropyl‐β‐, Methyl‐β‐, and 2‐Hydroxypropyl‐γ‐Cyclodextrin: Effect on Aqueous Solubility, Dissolution Rate, and Ataxic Activity in Rat

Complexation of Zolpidem with 2-Hydroxypropyl-␤-, Methyl-␤-, and 2-Hydroxypropyl-␥-Cyclodextrin: Effect on Aqueous Solubility, Dissolution Rate, and Ataxic Activity in Rat GIUSEPPE TRAPANI,1 ANDREA LATROFA,1 MASSIMO FRANCO,1 MARIA ROSARIA PANTALEO,1 ENRICO SANNA,2 FEDERICO MASSA,2 FRANCESCA TUVERI,2 GAETANO LISO1 1

Dipartimento Farmaco-Chimico, Facolta` di Farmacia, Universita` degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy

2

Dipartimento di Biologia Sperimentale, Cattedra di Farmacologia, Universita` di Cagliari, 09123 Cagliari, Italy

Received 22 December 1999; revised 27 January 2000; accepted 14 July 2000

The effect of some chemically modified cyclodextrins [namely, 2-hydroxypropyl-␤-, methyl-␤-, and 2-hydroxypropyl-␥-cyclodextrin (HP-␤-CD, Me-␤-CD, and HP-␥-CD, respectively)] on the aqueous solubility and dissolution rate of the hypnotic agent Zolpidem (ZP) was investigated. Solid complexes were prepared by freeze drying and characterized by infrared spectroscopy, X-ray powder diffraction, and differential scanning calorimetry. The solubility and dissolution rate of the drug were significantly improved by complexation with HP-␤-CD or Me-␤-CD. The structure of the inclusion complex ZP–HP-␤-CD in CH3COOD/D2O was investigated by 1H and 13C NMR spectroscopy, including NOE measurements. These measurements revealing a weak interaction between the tolyl moiety of the guest molecule and the HP-␤-CD cavity. The ataxic activity in rat was also investigated and it was found that ZP–HP-␤-CD and ZP–Me-␤-CD complexes showed almost 2-fold longer ataxic induction times than controls. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: ABSTRACT:

1443–1451, 2000

Keywords: Zolpidem; hydrophilic cyclodextrins; solid-state study; solubility study; NMR measurements; ataxic activity

INTRODUCTION Zolpidem (ZP), N,N-dimethyl-[2-(4-tolyl)-6methylimidazo[2,1-a]pyridin-3-yl]acetamide, is a drug with strong hypnotic and sedative actions. ZP is widely used for the treatment of insomnia and sleep disorders1 and is currently formulated as a hemitartrate salt in tablet form (Stilnox威), which has a somewhat limited oral bioavailability (∼70%). Because ZP is sparingly water soluble and possesses a slow dissolution rate, it may be thought that its dissolution in gastrointestinal fluids constitutes the rate-limiting step for the absorption process. Correspondence to: G. Trapani (Telephone: 039-0805442764; Fax: 039-080-5442724; E-mail: trapani@farmchim. uniba.it) Journal of Pharmaceutical Sciences, Vol. 89, 1443–1451 (2000) © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association

In a previous paper,2 we investigated the effect on the solubility and dissolution rate of ZP in solid dispersions and in physical mixtures with polyethylene glycol 4000 and 6000 (PEG 4000 and 6000, respectively). Regardless of an increase in solubility, its concentration remained below the limits (10 mg/mL) generally accepted for an optimal oral formulation. As a continuation of our study, we investigated the influence of ZP complexation with some chemically modified cyclodextrins (CDs) on drug solubility and dissolution behavior, the major goal being improvements to the solubility, dissolution rate, and oral bioavailability of ZP.3 We therefore investigated the complexation of ZP with hydrophilic CDs, both of hydroxypropylated [i.e., hydroxypropyl-␤-CD (HP-␤CD) and hydroxypropyl-␥-CD (HP-␥-CD)] and randomly methylated type (Me-␤-CD). Each complex was characterized by usual physicochemical

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methods, including Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray analysis. Phase solubility diagrams and intrinsic dissolution profiles of the complexes were constructed and comparatively evaluated. Furthermore, nuclear magnetic resonance (NMR) spectroscopic studies of the ZP–HP-␤-CD system were carried out to gain insights into the complexation mode of ZP. Finally, the ataxic activity in rats of these new ZP– CD formulations was also evaluated and compared with that of Stilnox威.

EXPERIMENTAL SECTION

vent and were linear (r2 > 0.998) over the range of concentrations of interest. Preparation of Solid ZP–CD Complexes In preparing the solid complex by a freeze-drying method, ZP (0.66 mmol) and equimolar quantities of the appropriate CD in 5 mL of deionized water were equilibrated. The mixture was stirred at room temperature for 4 days and filtered through a 0.22-␮m membrane filter, and the clear filtrate was subjected to freeze drying (Edwards model type 680 freeze-drier). Solid-State Study

Materials

Fourier Transform Infrared Spectroscopy

ZP was extracted from tablets of Stilnox威 purchased from a local drugstore as follows. Thirty Stilnox威 tablets were powdered in a mortar, and the powder was dissolved in 10% aqueous NaHCO3 (50 mL). The solution was transferred to a shake flask and extracted with ethyl ether (3 × 30 mL). The organic layer was dried (Na2SO4) and evaporated. The solid residue was the pure ZP as identified by spectroscopic methods (IR,1H NMR, and mass spectroscopy) and comparison with authentic sample. Reagents used for preparations of buffers were of analytical grade. 2-Hydroxypropyl-␤-cyclodextrin with a degree of substitution of 5.88 (calculated by 1H NMR4) was obtained as a gift from Roquette (Italy). 2-Hydroxypropyl-␥cyclodextrin (degree of substitution, 0.6) and methyl-␤-cyclodextrin (degree of substitution, 1.8) were kindly donated from Waker-Chemie. Fresh deionized water from an all-glass apparatus was used. The high-performance liquid chromatography (HPLC) mobile phase was prepared with HPLC-grade methanol. The HPLC analyses were performed with a Water Associates model 600 pump equipped with a Water 990 variable wavelength ultraviolet (UV) detector and a 20-␮L loop injection valve. For analysis, a reversed-phase Simmetry C18 (25 cm × 3.9 mm; 5 ␮m particles) column in conjunction with a precolumn insert was eluted with mixtures of methanol and deionized water (75:25). The flow rate of 1 mL/min was maintained. Quantification of the compounds was carried out by measuring the peak areas in relation to those of standards chromatographed under the same conditions. Standard calibration curves were prepared at a wavelength of 245 nm using methanol as the sol-

The FTIR spectra were obtained on a PerkinElmer 1600 FTIR spectrometer. Samples were prepared in KBr disks (2 mg sample in 200 mg KBr). The scanning range was 450–4000 cm−1 and the resolution was 1 cm−1.

X-ray Analysis X-ray powder diffraction (XRPD) patterns were recorded on a Philips PW 1800 powder X-ray diffractometer using Ni-filtered, CuK␣ radiation, a voltage of 45 kV, and a current of 25 mA.

Differential Scanning Calorimetry DSC curves were obtained by a Perkin-Elmer DSC 7, equipped with a thermal analysis (TA) automatic program. Aliquots of ∼5 mg of each sample were placed in an aluminum pan of 50 ␮L capacity and 0.1 mm thickness, press-sealed with a nonperforated aluminum cover of 0.1 mm thickness. An empty pan sealed in the same way was used as reference. Thermograms were measured by heating the sample from 30 and 300 °C at a rate of 10 °C/min, under a nitrogen flow of 20 cm3/min. Indium was used as standard for calibrating the temperature. Reproducibility was checked by running the sample in triplicate. Phase–Solubility Analysis Solubility measurements of ZP were carried out at a constant temperature (37 °C) using CD aqueous solutions at various concentrations. In these studies, the CD solutions were made with deionized water. A large excess of the hypnotic agent

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was added to 2 mL of the appropriate CD solution in screw-capped test tubes. The mixtures were mixed by vortexing for ∼5 min and shaken in a thermostatically controlled water bath shaker for 5 days. Then, an aliquot of aqueous phase of each mixture was transferred to a 10-mL glass syringe preheated at the appropriate temperature and filtered through a 0.22-␮m membrane filter (Millipore威 cellulose acetate) in thermostated test tubes. Next, ∼0.5 mL of the clear filtrate was appropriately diluted (1:100), and 20 ␮L of this last solution was analyzed by HPLC. All of the manipulations were made without the removal of the test tubes from the water bath, using thermostated pipettes and syringes. The apparent 1:1 stability constant (Kc) was estimated from the slope of the straight line of the phase–solubility diagram according to the following equation: Kc ⳱ slope/S0(1 − slope).5 The solubility value (S0) of ZP was determined previously in deionized water at the temperature of the experiment.

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mg of solid complex in 0.6 mL of 10% (v/v) CH3COOD in D2O. This mixture allows a suitable solubilization of the free drug for these experiments. Two-dimensional (2D) nuclear Overhauser enhancement spectroscopy (NOESY), correlation, and one-dimensional proton nuclear Overhauser (1D 1H NOE) experiments were carried out with the same solutions as used for 1H NMR. Samples were deaerated by bubbling N2 directly in the NMR tube. The 2D NOESY spectra were measured under the following conditions: sweep width, 3600 Hz; carrier frequency, 12 ppm; spin-lock field, 3 kHz; mixing time, 0.2 s; number scans, 64; pulse delay, 1.0; data matrix, 2 × 128 × 1 K. The 1D 1H NOE spectra were recorded under the following conditions: number of scans, 128; acquisition time, 2.72 s; pulse width, 2.90 ␮s; time domain, 32 K; spectral width, 6024 Hz. The NOE measurements were performed by irradiation of the H-3 signals of HP-␤-CD at 25 °C. The increments of the signals (NOE %) were evaluated with the NOE difference program.

Dissolution Study Dissolution experiments were carried out in triplicate with an Erweka DT dissolution test in deionized water at 37 °C by the paddle method at a rotation speed of 60 rpm. ZP alone and some complexes prepared as already reported were included in this study. Samples of each solid complex (previously sieved, 280 ␮m) equivalent to 50 mg of ZP were added to the dissolution medium (400 mL). At appropriate time intervals, 2 mL of the mixture were withdrawn and filtered through a 0.22-␮m membrane filter (Millipore威 cellulose acetate) in thermostated test tubes. The initial volume of dissolution medium was maintained by adding 2 mL of deionized water. About 1 mL of the clear filtrate, after appropriate dilution, was allowed to stand in bath at 37 °C until analyzed by HPLC. The injection volume was 20 ␮L. The results were computed with a standard calibration curve of the drug. NMR Measurements The 1H and 13C NMR studies on the solid (freezedried) ZP–HP-␤-CD complex were performed at 25 °C with Bruker AM 300 WB (300 MHz) and Varian XL 200 (200 MHz) spectrometers, respectively. The 1H –13C correlation experiments were performed on AVANCE 500 DRX (500 MHz) by using the invieagss i pulse sequence. The 1H and 13 C NMR spectra were obtained by dissolving 34

Pharmacology Male Sprague-Dawley rats (Charles River, Como, Italy) weighing 120–150 g were kept under a 12-h light–dark cycle at a temperature of 23 ± 2 °C and 65% humidity. On arrival at the animal facilities, there was a minimum of 7 days of acclimatization during which the animals had free access to food (until ∼12 h from starting experiments) and water. Rats (eight for each experimental group) received equimolar doses (10 mg/kg) of ZP as solid complexes by intragastric administration with an appropriate sized needle. Animals of the control group were administered ZP suspended in distilled water containing one drop of Tween 80 per 5 mL. The different CD-based formulations were solubilized, in equimolar concentrations of ZP (10 mg/kg), in distilled water with a drop of Tween 80 per 5 mL. The drop of surfactant was added to solution because it was present in the control ZP suspension. Rats (eight per each group) were food-deprived for 12 h before the experiment. Rats were observed for the following 60 min, and the time of ataxic induction, which was defined as the time from drug administration to the status of profound sedation characterized by motor incoordination of all four legs, was recorded. Ataxic effect was subdivided into the following three distinct levels of deepness: (i) “ataxia level 1”, when the animal shows motor incoordination of posterior legs; (ii) “ataxia level 2”, when motor incoor-

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Studies were performed to ascertain the interaction between ZP and CDs in the solid state and to check the degree of crystallinity of the ZP–CD systems. FTIR spectra of ZP, HP-␤-CD, Me-␤-CD, and corresponding complexes are shown in Figure 1. The spectrum of ZP shows a strong absorption band of amide carbonyl stretching at 1633 cm−1, whereas the spectra of complexes show a broad band at ∼1600 cm−1. The latter appears similar to

the band in the spectra of pure CDs, which should be attributable to water content of CDs. Figure 2 shows the XRPD patterns of ZP, HP␤-CD, Me-␤-CD, and corresponding complexes. The presence of several distinct peaks in the XRPD of ZP at a diffraction angle of 2␽ 9.54, 19.18, 23.28, 26.37, 26.96, and 33.51° reveals that the drug is present as a crystalline material. On the other hand, the XRPD spectra of pure CDs and complexes were characterized by the complete absence of any diffraction peak. These results indicate that ZP is no longer present as a crystalline material when complexed with CDs, but exists in the amorphous state. The thermograms of ZP, HP-␤-CD, Me-␤-CD, and corresponding complexes are shown in Figure 3. The DSC curve of pure ZP showed a single endothermic peak at 193° corresponding to the melting point of the drug. The DSC profiles of pure CDs and complexes exhibited a broad endothermic peak ranging from 60 to 140 °C, and in the CD-based formulations, the ZP melting peak was not observed.

Figure 1. FTIR spectra of ZP, HP-␤-CD, Me-␤-CD, and corresponding complexes: (a) HP-␤-CD; (b) Me-␤CD, (c) ZP–HP-␤-CD, (d) ZP–Me-␤-CD, (e) ZP.

Figure 2. RPD patterns of ZP, HP-␤-CD, Me-␤-CD, and corresponding complexes: (a) HP-␤-CD, (b) ZP–Hp␤-CD, (c) Me-␤-CD, (d) ZP–Me-␤-CD, (e) ZP.

dination involves both anterior and posterior legs; and (iii) “ataxia level 3”, when animals are unable to walk and lay on their abdomen, but no loss of the righting reflex was observed if the animals were laid on their backs. The statistical significance of differences in behavioral data was analyzed by the analysis of variance (ANOVA) test followed by Newman–Keuls post hoc test.

RESULTS AND DISCUSSION Solid-State Study

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Figure 3. DSC spectra of ZP, HP-␤-CD, Me-␤-CD, and corresponding complexes: (a) Me-␤-CD, (b) ZP–Me␤-CD, (c) HP-␤-CD, (d) ZP–HP-␤-CD, (e) ZP.

Phase–Solubility Analysis ZP is a weak base with a pKa of 6.2. The solubility of the free base is 250 ␮g/mL at 37 °C, a factor of 40 below the targeted solution concentration of 10 mg/mL generally accepted for optimal oral formulation. This target concentration is achieved with the currently marketed formulation (i.e., Stilnox威). It is well known, however, that salts of poorly water-soluble weak bases suffer from the potential of precipitation at pH values found in the gastrointestinal tract. Solubility experi-

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ments showed that the concentration of ZP in water is markedly affected by the presence of CDs (Table 1). Thus, 45% w/v HP-␤-CD and Me␤-CD solutions provided, respectively, 10.71 ± 1.07 and 9.52 ± 0.73 mg/mL contents of ZP, which correspond to 42 and 38 times increases in ZP concentration, respectively. On the other hand, a 30% w/v HP-␥-CD solution provided a 1.55 ± 0.01 mg/mL ZP content corresponding to a 6-fold increase in ZP water solubility, which is comparable to that previously observed for PEG-based formulations.2 In summary, we achieved the targeted solution concentration of 10 mg/mL by preparing complexes of ZP with HP-␤-CD and Me-␤CD only. The phase–solubility diagrams investigated using deionized water (pH 6.5) were linear in a wide range of CD concentrations and correspond to AL-type5 profiles (plots not shown). Because a slope of <1 characterizes such profiles of the phase–solubility diagrams, it was assumed that the solubility increase is due to the formation of a 1:1 complex. The apparent stability constants (K1:1; Table 2) were estimated from the slope of the straight line of the phase–solubility diagrams according to the following equation: K c ⳱ slope/S0(1− slope),5 where S0 is the solubility value of ZP in deionized water. As expected, the stability constant values with the two ␤-CDs were higher than that observed with ␥-CD (152 and 119 M−1for HP-␤-CD and Me-␤-CD, respectively, versus 27 M−1 for HP-␥-CD). Furthermore, the low apparent stability constants are indicative of a shallow interaction. Table 2 also reports the degree of ZP incorporation in the solid complexes, with the highest values occurring for HP-␤-CD and Me-␤-CD complexes (29.5 and 27 mg/g of complex, respectively).

Table 1. Effect of Various Concentrations of HP-␤-CD or HP-␥-CD and Me-␤-CD on Zolpidem Solubility (S) at 37 °C.a HP-␤-CD (% w/v)

S (mg/mL)

HP-␥-CD (% w/v)

S (mg/mL)

Me-␤-CD (% w/v)

S (mg/mL)

0.0 4.5 9 18 27 36 45

0.25 ± 0.04 1.38 ± 0.10 2.56 ± 0.19 4.68 ± 0.14 6.83 ± 0.44 8.77 ± 0.60 10.71 ± 1.07

0.0 5.6 9 11.9 17.8 23.7 30

0.25 ± 0.04 0.53 ± 0.02 0.67 ± 0.02 0.78 ± 0.05 1.00 ± 0.55 1.33 ± 0.08 1.55 ± 0.01

0.0 4.5 9 18 27 36 45

0.25 ± 0.04 1.33 ± 0.20 1.91 ± 0.36 4.20 ± 0.27 5.79 ± 0.61 7.97 ± 0.02 9.52 ± 0.73

a

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Table 2. Stability Constants and Degree of Incorporation of Zolpidem and HP-␤-CD or HP-␥-CD or Me-␤-CD Complexation in Deionized Water

C

Apparent Stability Constant K1:1 (M−1)a

Degree of Zolpidem Incorporation (mg/g)b

HP-␤-CD HP-␥-CD Me-␤-CD

152 (7) 27 (2) 119 (5.5)

29.5 (8) 5.6c 27 (10)

a Mean of three determinations; relative standard deviation (CV) values are reported in parentheses. b Mean of three determinations at 37 °C; relative standard deviation (CV) values are reported in parentheses. c Single determination.

Dissolution Study Because the effect of HP-␥-CD on the solubility of ZP was negligible, we did not include the corresponding complex in dissolution studies. The amounts of ZP used in dissolution experiments (50 mg or equivalent amount for complexes in 400 mL of medium) were below the saturation concentration, so sink conditions could be assumed. The results, expressed as the mean of three determinations, are shown in Figure 4. The dissolution data showed coefficient of variation (CV) values of <10%, but at times soon after dissolution begins,

Figure 4. Dissolution profiles of ZP alone, ZP–HP-␤CD, and ZP–Me-␤-CD. Data are the mean of three determinations.

a greater variability was observed (CV of 15– 20%).6 As can be seen in Figure 4, the pure ZP dissolution rate is very slow, with ∼58% of the drug being dissolved after 1 h; in contrast, the dissolution of complexes is effectively completed within 10 min. The observed Q10, Q30, and Q60 values (i.e., percent of dissolved ZP at 10, 30, and 60 min)7 were 28, 43, and 57% for the pure drug and 100% for ZP–HP-␤-CD and ZP–Me-␤-CD systems, respectively. Uekama and Stzejtli suggested mechanisms of increased dissolution rate by CD complexation.8,9 According to these suggestions, we believe that a decrease in the crystallinity of the drug and an increase in its solubility could be major factors determining the enhanced dissolution. NMR Measurements The ZP molecule contains two separate aromatic groups that may lead to both 1:1 and 1:2 complexation with HP-␤-CD. To ascertain the structure of the solid complex formed by ZP with HP-␤-CD, a 1H and 13C NMR study was carried out. The numbering of the H and C atoms of ZP for NMR interpretation is reported in Table 3 and Table 4, respectively. At first, we successfully assigned all the 1H NMR signals of ZP. Whereas the proton assignments for the methylene group and the tolyl and pyridine moieties were easily made, it was difficult to unambiguously assign the resonances of the methyl groups in the 1H NMR spectrum of ZP. This last issue was addressed by analyzing the 2D NOESY 1H NMR spectrum of ZP, which showed the presence of cross-peaks between the proton linked at C-5 and the methyl group at C-6, as well as between the protons of the methyl group at C-4⬘ and those of the nearest carbons in the benzene ring (C-5⬘ or C-3⬘). The chemical shifts for the protons of ZP both in the absence and presence of HP-␤-CD are summarized in Table 3 as ␦ free and ␦ complex, respectively. As can be seen, small changes in chemical shift (∼0.01–0.05 ppm) were generally observed. The largest downfield shifts on complexation were observed for the protons of the tolyl group. Next, to gain further structural information, a 13 C NMR spectroscopic study was also performed. In this regard, it is well known10,11 that 13C NMR chemical shifts are sensitive probes of molecular environments and they can be used to derive information on complexation. Because of the complexity of the 13C NMR spectrum, it was difficult to unambiguously assign all 13C signals of ZP, but

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Table 3. 1H Chemical Shifts Corresponding to Zolpidem in Absence and Presence of HP-␤-CD in CD3COOD/D2O (10% v/v)

Zolpidem Proton H-14 H-13 H-11 H-12 H-9 H-2⬘, H3⬘, H5⬘, H6⬘ H-7, H-8 H-5 a

Table 4. 13C Chemical Shifts Corresponding to Zolpidem in Absence and Presence of HP-␤-CD

Peak Multiplicity

␦free

␦complex

⌬␦a

2.0719 2.1409 2.6865 2.8851 3.9976

2.1261 2.1627 2.6936 2.9041 4.0259

0.0542 0.0218 0.0071 0.0190 0.0283

singlet singlet singlet singlet singlet

7.1132 7.5072 7.9135

7.1721 7.5429 7.9509

0.0589 0.0357 0.0374

double doublet multiplet singlet

⌬␦ ⳱ ␦complex − ␦free

Zolpidem Carbon

␦free

␦complex

⌬␦a

C-13 C-14 C-9 C-11 C-12 quaternary C ternary C C-2⬘, C-6⬘ C-3⬘, C-5⬘ C-10

19.695 22.937 30.627 38.205 39.826 113.284 118.943 130.464 132.485 171.474

20.379 20.411 N.D.b 38.185 39.774 113.299 119.193 130.722 132.465 171.301

0.684 −2.526 — −0.070 −0.062 0.015 0.250 0.258 −0.020 −0.173

b

zolpidem.

for some of them it was possible. The 13C chemical shifts and carbon assignments made for ZP as well as the 13C chemical shift modifications on complexation are shown in Table 4. Interestingly, when comparing 1D 1H with 13C NMR spectra, we noticed that whereas the proton signals of the methyl group on the pyridine moiety (H-13) resonate at lower fields than those of the tolyl group (H-14), the opposite situation was observed for the corresponding carbon (C-13 and C-14) signals. Assignment of the resonances of the methyl groups of ZP followed by a 2D 1H–13C correlation analysis clearly confirms the assignments made. On addition of HP-␤CD, a significant change in chemical shift (⌬␦ ⳱ −2.526 ppm) was observed for C-14. Taken together, the NMR results and the low value of the apparent stability constant for the ZP–HP-␤-CD complex led us to infer that ZP forms a shallow inclusion complex through interactions between the tolyl moiety of the ZP and HP-␤-CD, without any involvement of the pyridine moiety. To further support the aforementioned inclusion mode, 1D 1H NOE experiments14 were performed, which showed a small but significant increment (0.5%) in the H-14 proton signal by irra-

⌬␦ ⳱ ␦complex − ␦free Not detected.

a

zolpidem.

diation of the HP-␤CD H-3. Furthermore, in the 2D NOESY spectrum of the ZP–HP-␤-CD system, cross-peaks were observed between the H-14 protons of ZP and the H-3 proton of HP-␤-CD as well as between the H-14 protons of ZP and the methyl proton of the 2-hydroxypropyl group of HP-␤-CD (Figure 5). These cross peaks, although of low intensity, are significant and were not observed in the 2D NOESY spectrum of the drug alone. These data are in good agreement with the proposed inclusion mode involving a weak interaction between the tolyl moiety of the ZP molecule and the cavity of HP-␤-CD. Pharmacology To verify whether the increase in dissolution rates observed with CD based formulations may lead to differences in pharmacological effects, we investigated the ataxic action in rats following oral administration of ZP as suspension, CDbased formulations of ZP, and Stilnox威 in equimolar doses (10 mg/kg). Times of ataxic induction and duration following the intragastric administration of each formulation were recorded, and the results are summarized in Table

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Figure 5. The 2D NOESY spectrum of the ZP–HP-␤CD complex.

5. In these experiments, at the dose of ZP administered, rats displayed a marked sedation and ataxia that was characterized by motor incoordination involving all four legs; however, no loss of the righting reflex was observed if the animals were laid on their backs. As shown in Table 5, ataxic induction times subsequent to intragastric administration of ZP as CD complexes were longer than those of reference formulations, even though the duration of ataxia was not modified. Indeed, ZP–HP-␤-CD and ZP-Me-␤-CD complexes, in spite of their fast dissolution rates, show almost 2-fold longer ataxic induction times

Table 5. Evaluation of the Ataxic Effect in Rats of Zolpidem in the Presence of HP-␤-CD, Me-␤-CD, and HP-␥-CDa

Zolpidem Formulations

Time to Ataxia (min)

Duration of Ataxia (min)

Zolpidem, 10 mg/kg, po Stilnox威 ZP–HP-␤-CD ZP–Me-␤-CD ZP–HP-␥-CD

11.7 ± 2.4 13.6 ± 1.1 22.6 ± 1.4b,c 22.1 ± 0.3b 22.5 ± 9.5b

22.5 ± 5.4 27.5 ± 5.4 32.5 ± 7.3 29.4 ± 4.9 33.4 ± 6.4

a Zolpidem was suspended in distilled water with a drop of Tween 80 per 5 mL. The different CD-based formulations were solubilized, in equimolar concentration of zolpidem (10 mg/ kg), in distilled water with a drop of Tween 80 per 5 mL. b p < 0.05 versus Zolpidem- or Stilnox威-treated animals. c Rats treated with ZP–HP-␤-CD displayed, for the time period considered (1 h), a lower level of ataxia (level ataxia 2) with respect to the other groups of animals and never reached the status of “ataxia 3”. The time indicated for this group is thus relative to this lower level of ataxia.

than controls. Similar results have recently been obtained using ZP in solid dispersion with PEGs,2 which is somewhat surprising considering CDand PEG-based formulations were given as solutions, whereas the controls were administered as suspensions. Provided that in vitro dissolution data can be used to assess in vivo performance, our results may suggest that dissociation of ZP– HP-␤-CD and ZP–Me-␤-CD complexes, rather than their dissolution in gastrointestinal fluids, could be the rate-limiting factor in the gastrointestinal absorption of the drug.15 In other words, when ZP is complexed, its dissociation3,16 may be the rate-limiting step of the transfer across the intestinal membrane, resulting in a slow systemic absorption and pharmacokinetic features different from those of the free drug. This explanation may be consistent with our finding that rats treated with ZP–HP-␤-CD complex display a lower level of ataxia (ataxia level 2) and never the status of “ataxia 3” (Table 5). On the other hand, a similar behavior had been already observed by others evaluating CD complexes with the local anesthetic bupivacaine, the latter being characterized by affinity constants very close to those of ZP–HP-␤-CD and ZP–Me-␤-CD complexes.17,18 By comparison, Koizumi et al. found that complexation of barbiturates with ␤-CD considerably decreased induction times.19 It should be pointed out that no definitive conclusion can at present be drawn to explain the differences observed in ataxic induction times, especially considering the recent paper of Stella and coworkers20 on the mechanism of drug release from CD complexes. Further study is necessary to address this issue.

CONCLUSIONS The results of this study show that the dissolution rate of the ZP–HP-␤-CD and ZP–Me-␤-CD complexes are much faster than that of ZP alone as a consequence of the increased solubility and decrease in crystallinity caused by complexation. Results from NMR spectroscopic studies indicate that ZP interacts weakly with HP-␤-CD, giving an inclusion complex involving the tolyl moiety of the ZP. The pharmacological study demonstrates that ZP complexation with HP-␤-CD or Me-␤-CD results in longer ataxic induction times than those of the drug alone and Stilnox威, without significant variation of the duration of ataxia. In addition, comparison of in vivo data shows that the complexation of ZP leads to ataxic induction times

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EFFECT OF CYCLODEXTRINS ON ZOLPIDEM WATER SOLUBILITY AND DISSOLUTION IN RATS

longer than those previously observed by ourselves with PEG-based formulations.2 Finally, the results obtained could provide a rational basis for designing and developing a fast-dissolving and sustained-release form of the hypnotic agent ZP.

ACKNOWLEDGMENTS Thanks are due to Roquette Italia and Waker Chemie for their kind gifts of HP-␤-CD and HP␥-CD Me-␤-CD, respectively. Thanks are also due to Prof. Fanizzi for providing the AVANCE NMR facilities at CARSO (Cancer Research Center -Valenzano Bari) and to Dr. Di Masi (Dipartimento Farmaco-Chimico, Universita` di Bari) for skillful technical assistance in NMR studies. We thank Prof. F. Stasi and Dr. M. R. Provenzano (Universita` di Bari) for help in recording X-ray and DSC spectra, respectively. This work was supported by a grant from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST).

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