Inclusion Complexation of Propofol with 2-Hydroxypropyl-â-cyclodextrin. Physicochemical, Nuclear Magnetic ResonanceSpectroscopic Studies, and Anesthetic Properties in Rat

GIUSEPPE TRAPANI,* ANDREA LATROFA, MASSIMO FRANCO, ANGELA LOPEDOTA, ENRICO SANNA,† ANDGAETANO LISO

Contribution from Dipartimento Farmaco-Chimico, Facolta di Farmacia, Universita degli Studi di Bari, Via Orabona 4,70125 Bari, Italy, and Dipartimento di Biologia Sperimentale, Cattedra di Farmacologia, Universita di Cagliari,Via Palabanda 12, 09123 Cagliari, Italy.

Received May 2, 1997. Final revised manuscript received December 26, 1997.Accepted for publication January 7, 1998.

Abstract 0 An aqueous formulations of propofol 1 can be preparedby solubilizing it in the presence of 2-hydroxypropyl-â-cyclodextrin(HP-â-CD). This is potentially useful for parenteral administration ofthe drug. The aqueous solubility of 1 linearly increased as a functionof HP-â-CD concentration and showed features of an AL type diagram.Thermodynamic parameters were obtained by using the temperaturedependence of the stability constant at temperatures of between 25and 37 °C. The results indicate that complex formation is enthalpically,rather than entropically, driven and that it may involve van der Waals(dispersive) forces, rather than hydrophobic interactions. The structureof the inclusion complex propofol/HP-â-CD was investigated in D2O,using 1H and 13C NMR spectroscopy. These studies revealed thatthe whole aromatic ring, as well as part of the isopropyl groups of theguest molecule, is located inside the HP-â-CD cavity, while the hydroxygroup is located at the rim of the wider cavity end. The geometricalfeatures of the inclusion complex 1−HP-â-CD are confirmed by 1DNOE difference spectra and molecular modeling experiments. Theanesthetic activity in rat was investigated, and it was found that thereare significant differences in induction time and sleeping time between1 solubilized in the presence of HP-â-CD and the formulation currentlyused (Diprivan), which is a 1% w/v oil/water emulsion.

Introduction

Propofol (2,6-diisopropylphenol) 1 (Chart 1) was firstintroduced into clinical practice as an intravenous anes-thetic agent, characterized by a short recovery time eitherafter a single bolus injection or after infusion.1 It has thedesirable properties of rapid onset and offset of effectfollowing rapid iv administration and minimal accumula-tion on long-term administration.2 However, despite thesefavorable clinical properties, which account for propofol’spopularity in anesthesia, its neurochemical action mech-anism has still to be clarified. A number of studies haveindicated that γ-aminobutyric acidA (GABAA) receptorsrepresent a sensitive target for the action of this intrave-nous anesthetic as well as for other general anesthetics.3-6

Similarly to other anesthetic agents (alphaxalone, al-phadolone), 1 is characterized by a very limited aqueoussolubility and was at first formulated as a 1% watersolution containing 16% of the nonionic surfactant Cremo-phor EL. However, Cremophor EL has been implicated in

some adverse reactions when administered intravenously,including anaphylactoid reactions.1 Successively, the an-esthetic agent was formulated as 1% w/v of soya bean oil/water emulsion stabilized by purified egg phosphatide,which is the formulation currently used (Diprivan). Un-fortunately, lipid-based emulsions also suffer from severallimitations, including poor physical stability, the potentialfor embolism, and pain on injection.7 Furthermore, strictaseptic techniques must be maintained when handlingthese formulations since they contain no antimicrobialpreservatives and therefore can support rapid growth ofmicroorganisms.8 Because of these shortcomings, thedevelopment of a safer formulation for 1 as an alternativeto the one currently used should be considered of notableinterest in anesthesiology. A plausible way which mayobviate the need for either surfactants or emulsions is thedevelopment of an acceptable aqueous parenteral formula-tion of the anesthetic agent. In principle, a number ofmethods can be envisaged for this purpose. Among thesevarious approaches, an attractive possibility is representedby the use of natural and modified cyclodextrins. Inparticular, chemically modified cyclodextrins have beenextensively used to increase the solubility, dissolution rate,and bioavailability of poorly water-soluble drugs as wellas to increase the stability of labile drugs.9-12 In particular,2-hydroxypropyl-â-cyclodextrin (HP-â-CD) deserves specialattention since its favorable physicochemical and biologicalproperties have been widely demonstrated.13-15

Solubility experiments in our laboratory16 showed thatthe approach can improve the aqueous solubility of propofolby inclusion complexation with HP-â-CD, a finding whichmight be exploited to provide a safe parenteral formulationfor the anesthetic agent. However, besides the knowledgeof drug solubility, binding constant(s), and dilution effect,12

* Corresponding author. Tel: 39 80 5442764. Fax: 39 80 5442724.E-mail: trapani@ippo.uniba.it.

† Universita di Cagliari.

Chart 1

S0022-3549(97)00178-0 CCC: $15.00514 / Journal of Pharmaceutical Sciences © 1998, American Chemical Society andVol. 87, No. 4, April 1998 American Pharmaceutical AssociationPublished on Web 02/27/1998

an essential factor in the preparation and use of formula-tions based on cyclodextrin complexes is an understandingof the complexation mechanism and driving force involved.In the present work the thermodynamic parameters for the1-HP-â-CD complexation, and a deeper characterizationof the inclusion complex by NMR spectroscopy and molec-ular modeling study are reported. In addition, the anes-thetic potential in rat model of propofol in HP-â-CDcontaining aqueous solutions is also presented.

Experimental SectionMaterialss2-Hydroxypropyl-â-cyclodextrin (the degree of sub-

stitution, 1.0, was calculated by means of 1H NMR) and propofolwere purchased from Aldrich Chemical Co. Propofol was of extrapure reagent grade and chromatographically pure (TLC) samplesof distilled propofol were stored under nitrogen. Reagents usedfor the preparation of the buffers were of analytical grade. Freshdeionized water from all glass apparatus was used in the prepara-tion of all the solutions. HPLC mobile phase was prepared fromHPLC-grade methanol. High-performance liquid chromatography(HPLC) analyses were performed with a Water Associates Model600 pump equipped with a Water 990 variable wavelength UVdetector and a 20 µL loop injection valve. For analysis, a reversed-phase µBondapack C18 (30 cm × 3.9 mm; 10 µm particles) or areversed phase Simmetry C18 (25 cm × 3.9 mm; 5 µm particles)column in conjunction with a Guard-Pak or a precolumn insert,respectively, was eluted with mixtures of methanol and deionizedwater (8:2). The flow rate of 1 mL/min was maintained. Quan-tification of the compounds was carried out by measuring the peakareas in relation to those of standards chromatographed underthe same conditions. Standard curves were prepared at a wave-length of 270 nm using methanol as the solvent and were linear(r 2 > 0.998) over the range of concentrations of interest.Solubility StudiessSolubility measurements of propofol were

carried out at a constant temperature (25, 30, 37 °C) using variousHP-â-CD (4.05%, 8.1%, 16.2%, 24.3%, 32.4%, and 40.5% w/v)aqueous solutions. In these studies, the cyclodextrin solutionswere made using 0.05 M potassium phosphate buffer (pH 6.5). Alarge excess of the anesthetic agent was added to 2 mL of theappropriate HP-â-CD solution in screw-capped test tubes. Themixtures were vortexed for about 5 min and shaken in a thermo-statically controlled water bath shaker for 5 days. Then, analiquot of aqueous phase of each mixture was transferred to a 10mL glass syringe preheated at the appropriate temperature andfiltered through a 0.45 µm membrane filter (Millipore, celluloseacetate) in thermostated test tubes. Next, about 0.5 mL of theclear filtrate was collected, and after appropriate dilution (1:5) with0.05 M potassium phosphate buffer (pH 6.5), the test tubes wereallowed to stand at the appropriate temperature until analyzedby HPLC. The injection volume was 20 µL. All of the manipula-tions were done, except the removal of the test tubes from thewater bath, using thermostated pipets, syringes, and buffersolution. The apparent 1:1 stability constant (Kc) was estimatedfrom the slope of the straight line of the phase-solubility diagramaccording to the following equation: Kc ) slope/S0(1 - slope).17The solubility values (S0) of 1 were determined directly in 0.05 Mpotassium phosphate buffer (pH 6.5) at the temperature of theexperiment.Preparation of Solid Propofol-HP-â-CD ComplexsIn

preparing the solid 1-HP-â-CD inclusion complex by freeze-dryingmethod, propofol (0.418 g, 2.35 mmol) was equilibrated with HP-â-CD (3.24 g, 2.35 mmol) in 10 mL of deionized water. Theopalescent mixture was stirred at room temperature for 5 daysand filtered through a 0.45 µm membrane filter, and the clearfiltrate was subjected to freeze-drying (Edwards model type 680freeze-drier).NMRMeasurementss1H and 13C NMR spectra of solid 1-HP-

â-CD inclusion complex were taken at 25 °C on a Brucker Aspect3000 (300 MHz) or a Varian XL-200 (200 MHz) spectrometer,respectively. A 34 mg portion of solid complex obtained as abovewas dissolved in 0.6 mL of D2O or CH3OD for 1H NMR and 13CNMR spectra, respectively. The 13C NMR spectra were recordedin CH3OD to achieve a suitable solubilization of the free drug forthese experiments.The 1D 1H NOE experiments were carried out by using the

same solutions used for 1H NMR. Samples were deaerated by

bubbling N2 directly in the NMR tube. The spectra were recordedunder the following conditions: number of scans, 128; aquisitiontime, 2.72 s; pulse width, 2.90 µs; time domain, 32 K; spectralwidth, 6024 Hz. The NOE measurements were performed byirradiation of the H-3 and H-6 signals of HP-â-CD at 25 °C. Theincrements of the signals (NOE %) were evaluated with the NOEdifference program.Molecular ModelingsThe 1-HP-â-CD inclusion complex was

investigated using the SYBYL (version 6.0) software,18 runningon a Vax station 3100 connected to a PS 390 Evans-Sutherlandgraphic system, and the Tripos force field. The starting geometryof â-cyclodextrin was taken from the Cambridge crystallographicdata center; the hydroxypropyl derivative was built up by addingsix or seven hydroxypropyl groups to OH groups of â-cyclodextrinas a base molecule following literature suggestions.19 Propofol wasfitted into the cavity in an axial orientation. Several patterns ofsubstituent distribution were examined and each structure wasminimized. The energy minimization of the 1-HP-â-CD inclusioncomplex have been achieved by means of the program MAXIMIN2.Pharmacological StudiessMale Sprague-Dawley rats (175-

200 g) were kept under a 12 h light-dark cycle at a temperatureof 23 ( 2 °C and 65% humidity. Upon arrival at the animalfacilities there was a minimum of 7 days of acclimatization, duringwhich the animals had free access to food and water. Rats receivedan intravenous administration of the propofol given in equimolardoses (40 mg/kg) as HP-â-CD complex, as Diprivan, and as oil/water emulsion containing one drop of Tween 80 per 5 mL, thislast being sonicated prior the intravenous injection. For thispharmacological study, the 1-HP-â-CD inclusion complex wasprepared by considering the effect of various concentrations of HP-â-CD on propofol solubility previously reported.16 For iv admin-istration, animals were restrained in an appropriate plexiglasscage and a tail vein was used. The effects that were recordedincluded loss and reestablishment of righting responses, time ofanesthetic induction, and sleeping time. Anesthetic induction(sleep onset) was defined as the time from drug administration toloss of righting reflex, and the sleeping time was the time fromthe loss of the righting reflex until the animals were plantigradeon all four legs. The significance of differences in behavioral datawere analyzed utilizing analysis of variance (ANOVA) test.

Results and Discussion

We have already shown16 that the solubility of 1 inunbuffered deionized water is notably affected by thepresence of varying concentrations of HP-â-CD. Thus, a40.5% w/v HP-â-CD solution provided a 39.73 mg/mLcontent of 1 corresponding to a ∼266-fold increase insolubility of propofol compared to a simple aqueous system.The phase-solubility diagrams at various temperatures

now investigated in a 0.05 M potassium phosphate buffer(pH 6.5) were again linear in a wide range of HP-â-CDconcentrations and correspond to AL type profiles.17 Be-cause such profiles of the phase-solubility diagrams arecharacterized by a slope of less than 1, it was assumed thatthe solubility increase is due to the formation of a 1:1complex.17,20,21 The apparent stability constants (Kc) (Table1) were estimated from the slope of the straight line of thephase-solubility diagrams according to the equation Kc )slope/S0(1- slope), where the solubility values (S0) of 1 ina 0.05 M potassium phosphate buffer (pH 6.5) wereevaluated at 25, 30, and 37 °C. The thermodynamicparameters of complexation were obtained from the tem-perature dependency of the apparent 1:1 stability constantsshown by the inclusion complexes (Van’t Hoff plot) withinthe temperature range reported above and are also shownin Table 1. Complex formation is associated with a largenegative ∆G, a relatively large negative ∆H, and a smallnegative ∆S for in the system. Hence, as already observedfor similar inclusion complexes, the formation of thepropofol-HP-â-CD complex is mainly an enthalpicallyrather than an entropically driven process, and van der

Journal of Pharmaceutical Sciences / 515Vol. 87, No. 4, April 1998

Waals (dispersive) forces rather than hydrophobic interac-tions could be involved.11The inclusion complexation of 1 with HP-â-CD was also

confirmed by 1H and 13C NMR spectroscopy. In thefollowing text, for the sake of clarity, the atoms of HP-â-CD will be denoted with roman characters (H,C) and thoseof propofol with italicized characters (H,C). The chemicalshifts for the protons of 1 both in the absence and presenceof HP-â-CD are summarized in Table 2 as δ free and δcomplex, respectively. As can be seen, a significant down-field shift was observed for all protons of propofol in thepresence of HP-â-CD. Since, under the conditions em-ployed, only shift changes of the signals occur, it followsthat the complexation is a dynamic process in which a fastexchange exists between the free and the bound states.20-22

As for the proton resonances of HP-â-CD,22 we onlyexamined the chemical shifts of those protons located insidethe cavity and close to the large rim (H-3 and H-5) as wellas those located at the rim of the narrow side (H-6 andmethyl protons) (Table 3). In the presence of 1, each ofthese signals was shifted upfield (Table 3). The modifica-tions observed for these HP-â-CD protons, although oflower magnitude than those observed for the guest mol-ecule, are also indicative of the inclusion occurrence. InFigure 1, four different inclusion modes for the 1-HP-â-CD complex accounting for the observed shift changes arereported. For modes I and II, where the propofol moleculeis set deep within the HP-â-CD cavity, the downfield shiftof all signals attributable to 1 in the 1H NMR spectrathrough inclusion complexation is expected. On the otherhand, for modes III and IV, where the phenol compound ispartially located inside the cavity, the observed downfieldshift of all propofol signals may be explained by assumingthat the more stable conformations of the guest moleculeare those in which the alkyl protons of the isopropyl group(i.e. H-5 H-6) are included into the cavity. The choice ofthe correct inclusion mode followed from 13C NMR and 1D1H NOE studies. In this regard, it is well-known23 that13C NMR chemical shifts are sensitive probes of molecularenvironments and can be used to derive information on

complexation. For the guest molecule, the C-1, C-2, C-3,C-4, and C-5 carbons showed upfield shifts (Table 4), whilethe methyl carbon C-6 shifted in the opposite direction.Hence, the environment of the methyl carbons should bedifferent from that of other carbons. These findingssuggest that the phenolic hydroxy and methyl groupsshould be located outside the HP-â-CD cavity. On the otherhand, the rank order of ∆δ observed (Table 3) in 1H NMRfor the HP-â-CD protons directed toward the interior, i.e.,H-5 > H-6 > H-3, seems to be in good agreement with theassumption that the aromatic ring of the propofol moleculeis located completely inside the cavity. In fact, it isexpected that the aromatic moiety of the guest shouldinduce an upfield shift on the HP-â-CD protons located inthe proximity (i.e. H-5 and H-6) higher than the shiftinduced on the H-3 proton. These results, taken together,suggest that the aromatic ring is located completely insidethe cavity and close to the wider rim of the hollow cone ofcyclodextrin. Consistent with this hypothesis, a significantNOE effect was observed between the H-6 or H-3 protonsof 1 and the H-3 of HP-â-CD (Table 5). Furthermore, asignificant NOE effect was also observed between the H-4of propofol and the H-6 proton of HP-â-CD (Table 5). Onthe other hand, the rank order of the NOE % found is ingood agreement with the proposed inclusion mode III(Figure 1). Hence, on the basis of 1H and 13C NMR

Table 1sStability Constants and Thermodinamic Parameters of Complexation between Propofol and HP-â-CD in Phosphate Buffer (pH ) 6.5)

temp, °Cmolar solubility in phosphatebuffer (pH ) 6.5) 0.05 Ma

apparent stabilityconstant K1:1 (M-1)b ∆G°25°C (kJ mol-1) ∆H° (kJ mol-1) ∆S°25°C (J mol-1)

25 6.18 × 10-4 (8.5) 3439 (2) −20.17 −26.65 −21.7530 6.90 × 10-4 (10) 2942 (7.8)37 6.96 × 10-4 (1.6) 2228 (7.2)

a Mean of six determinations. Relative standard deviation (CV) values are reported in parentheses. b Mean of three determinations. Relative standard deviation(CV) values are reported in parentheses.

Table 2s1H Chemical Shifts Corresponding to Propofol in theAbsence and Presence of HP-â-CD

propofol proton δfree δcomplex ∆δ a peak multiplicity

H−3 6.571 6.901 +0.330 doubletH−4 6.438 6.686 +0.248 tripletH−5 2.567 3.124 +0.557 septetH−6 0.741 1.058 +0.317 doublet

a ∆δ ) δcomplex − δfree propofol.

Table 3s1H Chemical Shifts Corresponding to HP-â-CD in theAbsence and Presence of Propofol

HP-â-CD proton δfree δcomplex ∆δ a peak multiplicity

H−3 3.850 3.811 −0.049 multipletH−5 3.546 3.409 −0.137 multipletH−6 3.706 3.605 −0.101 multipletMethyl 0.985 0.951 −0.034 doublet

a ∆δ ) δcomplex − δfree HP-â-CD.

Figure 1sComplexation modes for the inclusion complex propofol−HP-â-CD.

Table 4s13C Chemical Shifts Corresponding to Propofol in theAbsence and Presence of HP-â-CD

propofol carbon δfree δcomplex ∆δ a

C−6 22.941 23.041 +0.100C−5 27.256 26.935 −0.321C−4 120.925 120.636 −0.289C−3 123.462 123.154 −0.308C−2 136.198 135.627 −0.571C−1 151.073 150.745 −0.328

a ∆δ ) δcomplex − δfree propofol.

516 / Journal of Pharmaceutical SciencesVol. 87, No. 4, April 1998

spectroscopy, it may be hypothesized that all the protonsof the aromatic ring, as well as some of those in theisopropyl groups, are located inside the hydrophobic HP-â-CD cavity, while the solvated hydroxy group is locatedat the rim of the wider cavity end where little or nodesolvation is needed upon complexation (mode III inFigure 1). Similar inclusion modes with the hydroxy groupat the wide cavity end were found for other phenolderivatives investigated.23,24 The proposed inclusion modeIII is also supported by computer-aided molecular modelingstudies. In Figure 2, a minimized computer-generatedstructure of propofol-HP-â-CD complex is depicted; thehydroxyl and methyl groups pushed outside the HP-â-CDcavity are clearly shown. The reported SYBYL-generatedmolecular model of the propofol-HP-â-CD inclusion complexcorresponds to four hydroxypropyl groups located at thelevel of primary OH groups at the rim of the narrow side(relative position of substituted glucoses: 1-3-5-6) andtwo hydroxypropyl groups located at the level of secondaryOH groups at rim of the wider side (relative position ofsubstituted glucoses: 3-7). Similar results were alsoobtained by locating seven hydroxypropyl groups on pri-mary and secondary OH groups of glucoses.The next aspect of these studies was to examine some

pharmacological properties of the potential HP-â-CD-basedformulation of 1. In this regard, it is interesting to pointout that recent studies aimed at evaluating the centralactions of propofol in HP-â-CD containing aqueous solu-tions in rabbits demonstrated that the intravenous admin-istration of propofol given in equimolar doses (15 mg/kg)both as cyclodextrin complex and as Diprivan did not showany differences in onset, duration, and maximum actionbetween the two dosage forms.25,12 On the other hand, ina previous investigation by Brewster,26 it was concludedthat the intravenous administration of 1 in HP-â-CDcontaining aqueous solutions in rat at doses ranging from5.5 to 22 mg/kg produced a dose response with a graduallyincreasing sleeping time from about 1 min to approximately15 min, but no comparison in anesthetic activity betweena HP-â-CD solution versus the commercial oil/water emul-sion (Diprivan) was made. Moreover, a recent studyexamined whether in rat the cardiovascular profile of 1dissolved in HP-â-CD differed from that measured forDiprivan, but once again no comparison in anestheticactivity was made.27 Therefore, we considered it to be ofinterest to investigate whether there are any differencesin terms of onset and duration of action in rat among thefollowing formulations: 1-HP-â-CD complex in water,Diprivan, and 1 as oil/water emulsion containing Tween80. The availability of these data should allow for a bettercomparison between the two animal models studied so far.As illustrated in Table 6, induction time after intravenousadministration of 1 as HP-â-CD complex was shorter thanthat observed when employing Diprivan. The differencesobserved in sleeping time are also significant. Data fromthis pharmacological paradigm, in fact, suggested that 1as an HP-â-CD complex produces almost 22% longersleeping time than Diprivan. Although to a lesser extent,

similar advantages in terms of induction time and sleepingtime were observed for propofol as an oil/water emulsioncontaining Tween 80 in comparison to Diprivan. Appar-ently, the animals did not feel pain on injection with thecyclodextrin formulation.The finding that in rabbit 1 as Diprivan and as an HP-

â-CD complex are practically indistinguishable pharma-cologically can be related to interspecies variations. Hence,to draw definitive conclusions concerning the advantagesof 1-HP-â-CD over Diprivan administration, further stud-ies utilizing these formulations on other animal models arerequired.In conclusion, the development of a safe formulation for

Table 5sNOE % Registered for the Propofol Protons by Irradiation ofH-3 and H-6 of HP-â-CD

NOE %

propofolproton

irradiation ofH−3/HP-â-CD

irradiation ofH−6/HP-â-CD

H−3 6.9 3.9H−4 5.8H−5 2.6H−6 8.5

Figure 2sComputer-generated model for the inclusion complex propofol−HP-â-CD. Dotted lines represent hydroxypropyl groups. (a) Top view. (b) Sideview.

Journal of Pharmaceutical Sciences / 517Vol. 87, No. 4, April 1998

a propofol alternative to Diprivan should be considered ofnotable interest in anesthesiology. Our study suggests thatpotentially acceptable parenteral formulations for 1 can beprepared by solubilizing the drug in aqueous 2-hydroxy-propyl-â-cyclodextrin (HP-â-CD) solution. The thermody-namic parameters for this complexation indicate that thisprocess is mainly enthalpically driven, and that van derWaals (dispersive) forces could be involved. The structureof the inclusion complex 1-HP-â-CD in D2O was deducedvia 1H and 3C NMR spectroscopy as well as by computer-aided molecular modeling studies. It may be hypothesizedthat the protons of the aromatic ring and isopropyl groupsare located inside the hydrophobic HP-â-CD cavity, whilethe solvated hydroxy group is located at the rim of thewider cavity end. In contrast to observations previouslymade in rabbit, pharmacological evaluations now per-formed in rat indicate that there are significant differencesin induction time and sleeping time between 1 solubilizedin the presence of HP-â-CD and the formulation currentlyused (Diprivan).

References and Notes1. Langley, M. S.; Heel, R. C. Propofol: A Review of Its

Pharmacodynamic and Pharmacokinetic Properties and Useas An Intravenous Anaestetic. Drugs 1988, 33, 334-372.

2. Dutta, S.; Matsumoto, Y.; Gothgen, N. U.; Ebling, W. F.Concentration-EEG Effect Relationship of Propofol in Rats.J. Pharm. Sci. 1997, 86, 37-43.

3. Collins, G. G. S. Effect of the Anaesthetic 2,6-Diisopropyl-phenol on Synaptic Transmission in the Rat Olfactory CortexSlices. Br. J. Pharmacol. 1988, 95, 939-949.

4. Concas, A.; Santoro, G.; Serra, M.; Sanna, E.; Biggio, G.Neurochemical Action of the General Anestetic Propofol onthe Chloride Ion Channel Coupled with GabaA Receptors.Brain Res. 1991, 542 , 225-232.

5. Hales, T. G.; Lambert, J. J. The Action of Propofol onInhibitory Amino acid Receptors of Bovine AdrenomedullaryChromaffin Cells and Rodent Central Neurons. Br. J. Phar-macol. 1991, 104 , 619-628.

6. Lin, L. H.; Chen, L. L.; Zirrolli, J. A.; Harris, R. A. GeneralAnesthetics Potentiate γ-Aminobutyric AcidA Actions onγ-Aminobutyric AcidA Receptors Expressed by XenopusOocytes: Lack of Involvement of Intracellular Calcium. J.Pharmacol. Exp. Ther. 1992, 266, 569-578.

7. Prankerd, R. D.; Stella, V. J. Use of Oil-in-Water Emulsionsas a Vehicle for Parenteral Drug Administration. J. Parent.Sci. Technol. 1990, 44, 139-149.

8. Bennett, S. N.; McNeil, M. M.; Bland, L. A.; Arduino, M. J.;Villarino, M. E.; Perrotta, D. M. Postoperative InfectionsTraced to Contamination of an Intravenous Anesthetic,Propofol. New Engl. J. Med. 1995, 333, 147-154

9. Szejtli, J. Cyclodextrins in Drug Formulations. Pharm.Technol. Int. 1991, 3(2), 15-22 and 1991, 3 (3), 16-24.

10. Uekama, K.; and Otagiri, M. Cyclodextrins in Drug CarrierSystems (Review) CRC Crit. Rev. Ther. Drug Carrier Syst.1987, 3, 1-40.

11. Loftsson, T.; Brewster, M. Pharmaceutical Applications ofCyclodextrins. 1. Drug Solubilization and Stabilizazion. J.Pharm. Sci. 1996, 85, 1017-1025.

12. Rajewski, R. A.; Stella, V. J. Pharmaceutical Applications ofCyclodextrins. 2. In Vivo Drug Delivery. J. Pharm. Sci. 1996,85, 1142-1169.

13. Pitha, J.; Milecki, J.; Fales, H.; Pannell, L.; Uekama, K.Hydroxypropyl-â-cyclodextrin: Preparation and Character-ization; Effects on Solubility of Drugs. Int. J. Pharm. 1986,29, 73-82.

14. Brewster, M.; Estes, K.; Bodor, N. An Intravenous ToxicityStudy of 2-Hydroxypropyl-â-cyclodextrin, a Useful DrugSolubilizer, in Rats and Monkeys. Int. J. Pharm. 1990, 59,231-243.

15. Irie, T; Uekama, K. Pharmaceutical Applications of Cyclo-dextrins. III. Toxicological Issues and Safety Evaluation. J.Pharm. Sci. 1997, 86, 147-162.

16. Trapani, G.; Lopedota, A.; Franco, M.; Latrofa, A.; Liso, G.Effect of 2-Hydroxypropyl-â-cyclodextrin on the AqueousSolubility of the Anaesthetic Agent Propofol (2,6-Diisopro-pylphenol). Int. J. Pharm. 1996, 139, 215-218.

17. Higuchi, T.; Connors, K. A. Phase Solubility Techniques. Adv.Anal. Chem. Instrum. 1965, 4, 117-212.

18. SYBYL Molecular Modelling Software Version 6.0; TriposInc., St. Louis, MO.

19. Mura, P.; Bettinetti, G.; Melani, F.; Manderioli, A. InteractionBetween Naproxen and Chemically Modified â-Cyclodextrinsin the Liquid and Solid State Eur. J. Pharm. Sci. 1995, 3,347-355.

20. Moyano, J. R.; Arias-Blanco, M. J.; Gines, J.M.; Rabasco, A.M.; Perez-Martinez, J. I.; Mor, M.; Giordano, F. Nuclearmagnetic resonance investigations of the inclusion complex-ation of gliclazide with â-cyclodextrin. J. Pharm. Sci. 1997,86, 72-75.

21. Djedaini, F.; Perly, B. NMR of Cyclodextrins, derivatives andinclusion compounds. In New Trends in Cyclodextrins andDerivatives; Duchene, D., Ed. Editions de Sante:, Paris, 1991;pp 217-246.

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Acknowledgments

The authors thank Dr. G. Carbonara for his assistance in themolecular modeling study. This work was supported by a grantfrom Ministero dell’ Universita e della Ricerca Scientifica eTecnologica (MURST) and from Consiglio Nazionale delle Ricerche(CNR).

JS970178S

Table 6sEffect of Intravenous Administration of Different PropofolFormulations on Loss of Righting Reflex and Sleeping Time in Rat

propofolformulations

loss of rightingreflex (s)

sleeping time(min)

HP-â-CD complex 122 ± 10 21.10Diprivan 315 ± 15a 17.25oil/water emulsion containing

Tween 80135 ± 10 19.5

a p < 0.05, statistically significant compared to Diprivan-treated animals(ANOVA). Values represent the mean ± SEM from 10 to 15 rats.

518 / Journal of Pharmaceutical SciencesVol. 87, No. 4, April 1998