International Journal of Pharmaceutics 165 (1998) 57–68

An econazole b-cyclodextrin inclusion complex: an unusualdissolution rate, supersaturation, and biological efficacy example

Morten Pedersen a,*, Simon Bjerregaard b, Jette Jacobsen b,Annette Rommelmayer Larsen b, Alex Mehlsen Sørensen c

a PEDCY, Frejas6ej 125, DK-7620 Lem6ig, Denmarkb Department of Pharmaceutics, The Royal Danish School of Pharmacy, 2 Uni6ersitetsparken, DK-2100 Copenhagen O, Denmark

c Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, 2 Uni6ersitetsparken,DK-2100 Copenhagen O, Denmark

Received 4 August 1997; received in revised form 7 October 1997; accepted 5 December 1997

Abstract

Genuine cyclodextrin inclusion complexes of the antimycotic econazole and b-cyclodextrin had higher antimycoticactivity than a physical mixture of econazole and b-cyclodextrin. Surprisingly, the econazole dissolution rate from thephysical mixture was higher than the dissolution rate from the inclusion complex. The improved antimycotic activityof the inclusion complex might be due to the superior ability of the complex to cause econazole supersaturation. Anew procedure was applied to disclose the drug supersaturation. The genuine inclusion complex molar ratioeconazole:b-cyclodextrin 2:3 gave rise to more hemolysis than the corresponding physical mixture. Toxicity testing ona human buccal epithelium in vitro model — based on TR146 cells — showed that the physical mixture was moretoxic than the inclusion complex when TR146 cell mortality was evaluated. Neither measurement of the transepithe-lial electrical resistance of TR146 cell layers exposed to either the physical mixture or the inclusion complex noranalysis of the protein liberation from the TR146 cells during exposure revealed any differences between the twocompositions. © 1998 Elsevier Science B.V. All rights reserved.

Keywords: Antimycotic; Biological response; Cell culture TR146; Cyclodextrin; Dissolution rate; Erythrocytes;Inclusion complex; Solubility diagram; Supersaturation; Toxicity

1. Introduction

Cyclodextrins are cyclic oligosaccharides whichare able to form complexes with lipophilic drugs,thus changing their physicochemical and biophar-maceutical properties.* Corresponding author. Tel.: +45 97821321.

0378-5173/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S0378-5173(97)00431-6

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–6858

Isolation of genuine b-cyclodextrin complexesof the lipophilic antimycotic drugs, econazole andmiconazole has been reported (Pedersen et al.,1993a; Pedersen, 1994). Compared with the puredrugs and with physical mixtures of drug andb-cyclodextrin, the genuine complexes showed im-proved antimycotic activity in vitro.

The overall aim of the present paper was tostudy the antimycotic effect of the econazole b-cyclodextrin complex in more detail. A newlyreported method was included to disclose possiblesupersaturation episodes during dissolution ratetesting of the econazole cyclodextrin inclusioncomplex (Pedersen, 1997).

In addition, a preliminary toxicity testing of theinclusion complex was carried out. Erythrocytesand a TR146 cell culture, a human buccal cellline, were employed.

2. Experimental

2.1. Materials

Econazole nitrate and b-cyclodextrin were pur-chased from Sigma (St. Louis, MO). Econazolebase was prepared from econazole nitrate as de-scribed previously (Pedersen et al., 1993a). Pep-tone and yeast extract were purchased from Difco(Detroit, MI). Candida albicans PF 1383 88 fromStatens Seruminstitut (Copenhagen, Denmark)was applied as test organism.

Dulbecco’s modified Eagle medium, gentamicinand other human cell culture media were obtainedfrom Gibco BRL (Paisley, UK). Heat-inactivatedfetal calf serum was purchased from Sera-Lab(Sussex, UK). The continuous cell line TR146,derived from a human neck mode metastasis orig-inating from a buccal carcinoma (Rupniak et al.,1985), was kindly provided by Imperial CancerResearch Technology (London, UK). Falcon cellculture inserts (polyethylene terephthalate, 1.6×106 pores/cm2, pore diameter 0.45 mm, growtharea 4.6 cm2) and Falcon 6-well culture plates(tissue culture treated polystyrene) were from Bec-ton Dickinson Labware (NJ).

2.2. Methods

2.2.1. Antimycotic acti6ity in a fluid mediumCultures of Candida albicans PF 1383 88 were

grown at 3791°C in sterile glucose, peptone,yeast extract growth medium (Pedersen and Rass-ing, 1990), adjusted to pH 7.5 with sterile 0.5 Mdisodium hydrogen phosphate buffer. At timezero 50 ml medium was inoculated with approxi-mately 5×105 Candida albicans cells per ml. A1-ml sample was taken from each flask containinginoculated broth to count the initial number ofcells. Pure drug, b-cyclodextrin, inclusion com-plex or physical mixtures of drug and b-cyclodex-trin were added to the broth. The compoundswere sieved through a 180-mm sieve just beforethe addition.

Samples were taken during a 24-h experimentalperiod. The fungal viability was estimated byplating 100 m l of appropriate dilutions of thesamples on agar plates. Colony counting wasperformed after incubation at 33°C for varioustime periods.

2.2.2. Solubility diagramsSolubility measurements were carried out as

described by Higuchi and Connors (1965). To 10ml of sterile Candida albicans microbial growthmedium, pH 7.5, temperature 3791°C, contain-ing various concentrations of b-cyclodextrin, 10mg of econazole nitrate were added. After ap-proximately 2 weeks, the suspensions were filteredthrough 0.2-mm Sartorius cellulose acetate mem-brane filters. The concentration of econazole inthe filtered samples was analyzed by an HPLCmethod.

2.2.3. Econazole b-cyclodextrin preparationsAbout 1 l ammonium phosphate buffer solution

0.05 M pH 7.1 containing 18.0 g b-cyclodextrinand 1.00 g econazole nitrate was shaken at 22°Cfor 10 days in a bottle with a screw cap. Duringthe period samples were taken from the bottle andthe solid phase was analyzed by differential scan-ning calorimetry to ensure that the econazolenitrate was completely transformed to the econa-zole b-cyclodextrin inclusion complex. After thetransformation was complete, the solid complex

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–68 59

was isolated by paper filtration. The complexwas washed with a few millilitres of water fol-lowed by drying at 60°C for 3 days.

Physical mixtures of b-cyclodextrin andeconazole or econazole nitrate were prepared bygently mixing in a mortar.

2.2.4. Differential scanning calorimetryA Perkin Elmer DSC7 was used. It was

equipped with a Perkin Elmer TAC/PC Instru-ment Controller and Perkin Elmer multitaskingsoftware. Closed aluminum pans were applied.The scan speed was 10°C/min. and nitrogen wasused as carrier gas. The sample size was in therange 2–5 mg.

2.2.5. X-ray powder diffraction analysisX-ray powder diffraction patterns were

recorded with a Guinier, XDC 700 IRDABpowder diffractometer using a Philips PW 1720X-ray generator. Cr Ka radiation was applied.

2.2.6. HPLC methodsEconazole concentrations were measured by a

reversed phase HPLC method (Pedersen et al.,1993a). Solid econazole preparations were dis-solved in dimethyl sulfoxide before the analysis.

The concentration of b-cyclodextrin in the mi-crobiological growth medium was determined bya reversed phase HPLC method. The eluent wascomposed of 15% methanol and 85% deionizedwater, the column was a Merck Lichrosorb 10018RP (4×125 mm) equipped with a Lichrosorb100 RP18 guard column. A Merck refractive in-dex detector was applied. The detection limit fora 20-m l loop was 0.5 mg/ml. The correlationcoefficient was 1.000 for the concentration range0.5–16 mg/ml.

2.2.7. Determination of water contentAbout 25.0 mg solid econazole b-cyclodextrin

complex was dissolved in methanol:dimethyl for-mamide 1:1. Afterwards the solution was titratedautomatically with Hydranal composite 5 (Riedelde Haen) on a Methrom 701KF Titrino (KarlFischer titration).

2.2.8. Dissolution testingThe dissolution testing of the drugs, physical

mixtures and inclusion complex was carried outusing the same experimental conditions as dur-ing the test of the antimycotic effect. In com-parison with the solubility of econazole in themicrobiological growth medium, excessiveamounts of econazole were added. That is, pos-sible drug supersaturation phenomena would bedisclosed. Samples were taken from the dissolu-tion medium and immediately filtered throughSartorius cellulose acetate 0.2-mm membranefilters. To avoid a possible precipitation ofeconazole after the filtration, one part of thefiltrate was mixed with three parts of dimethylsulfoxide. The mixtures were analyzed for econa-zole, while the neat filtrates were analyzed forb-cyclodextrin. Regarding the dissolutionmedium not filtered through Sartorius filters,precipitates were in some cases isolated by filtra-tion through paper filters. The precipitates werewashed with a few drops of water, dried andanalyzed.

2.2.9. HemolysisErythrocytes were separated by centrifugation

of citrated human blood at 1500×g for 10 minand washed three times with isotonic phosphatebuffer, 154 mM sodium chloride, 10 mM phos-phate, pH 7.4. The erythrocytes were resus-pended in the buffer to give a hemotocrit valueof 5% (Ohtani et al., 1989). Buffer 3.8 ml and0.2 ml erythrocyte suspension (5%) were mixed.The test substances, econazole, econazole nitrate,cyclodextrin inclusion complexes, physical mix-ture of cyclodextrin and econazole or cyclodex-trin alone were added as solid compounds attime zero. The samples were incubated in a 37°Cwater bath. Afterwards the samples were cen-trifuged for 3 min at 2000×g. The absorbanceof the supernatant was measured at 543 nm. A100% hemolysis value was obtained by mixing0.2 ml erythrocyte suspension and 3.8 ml dis-tilled water, instead of buffer, and incubatingthe sample at 37°C and measuring the 543 nmabsorbance of the supernatant (Ohtani et al.,1989).

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–6860

2.2.10. TR146 cell cultureTR146 cells were incubated and maintained in

25-cm2 T-flasks at 37°C in a 98% relative humid-ity atmosphere of 5% CO2/95% air. The culturemedium consisted of Dulbecco’s modified Eaglemedium supplemented with 10% heat-inactivatedfetal calf serum, 50 mg/ml gentamicin, and 0.2mg/ml p-hydroxybenzoic acid n-butyl ester. Fur-ther details concerning maintenance and seedingof TR146 cells were as described previously (Ja-cobsen et al., 1995).

The cell seeding density on the Falcon filterswas 2.4×104 cells/cm2. Of the culture mediummentioned above, 2.5 ml and 2.0 ml were addedto the apical and basolateral side, respectively.The culture medium was changed three times aweek. The cells were grown for 35 days on thefilters at the temperature and atmosphere men-tioned above before they were used for toxicity(trans-epithelial electrical resistance) studies.

2.2.11. Transepithelial electrical resistanceThe integrity of the TR146 cell layers on Fal-

con filters was evaluated before and during thecell culture toxicity study by measuring the electri-cal resistance (R; V) of the layers. Afterwards thetransepithelial electrical resistance was calculated(TEER; V cm2).

The measurement of R was carried out at 24°Capplying an Endohm-24 connected to an epithelialvoltohmmeter (EVOM), World Precision Instru-ments (FL). The Falcon filter inserts covered withcells and containing 2.5 ml Dulbecco’s modifiedEagle medium and the test substance were trans-ferred to the Endohm-24 which contained 4.5 mlmedium. R was recorded and TEER calculatedaccording to the equation:

TEER= (R(insert with TR146)−R(insert without TR146))×A

where A is the surface area of the filters, 4.6cm2.

2.2.12. Protein determination and TR146mortality

TR146 cells were grown to confluence in 25-cm2

T-flasks as described by Jacobsen et al. (1995).The growth medium was removed, and the flasks

were rinsed with 5×9 ml Hanks’ buffer. After-wards 10 ml Hanks’ buffer and the test substancewere added. The flasks were placed on an orbitalplate shaker at room temperature and 2-ml sam-ples were withdrawn after 30 and 240 min andfrozen until the protein determination was carriedout. After the last sampling, the flasks were rinsedwith 2×5 ml Hanks’ buffer and the cells wereexposed to 3 ml staining solution, Trypan BlueSolution 0.4% diluted 1:1 with water (Sigma Cata-logue 1530-1531, 1992). The TR146 mortality wascalculated by counting dead cells (stained blue)and total number of cells in three areas of theflasks using an inverted light microscope and agraticule (Jacobsen et al., 1995).

The protein concentration analysis was per-formed according to Lowry et al. (1951) measur-ing the absorbance at 280 nm and using alkalinecopper reagent and Folin and Ciocalteu’s phenolreagent. Bovine serum albumin was applied asstandard.

3. Results and discussion

3.1. Composition of inclusion complex

The econazole b-cyclodextrin complex was pre-pared, isolated and dried as described (n=3). Thedifferential scanning calorimetry indicated thatthe drug was completely converted to the complex(Fig. 1). That is, no peak from melting of freeeconazole was present (melting point 87°C). Be-fore equilibration was established, samples takenfrom the preparation bottles probably containedsome free econazole because a differential scan-ning calorimetry peak just below 87°C waspresent. Regarding the complex isolated afterequilibration, the X-ray powder diffraction pat-tern did not contain peaks from either free econa-zole or free econazole nitrate (Fig. 2).

According to the Karl Fischer titration, thewater content of the inclusion complex after dry-ing was 6.2% (n=3, S.D.=0.17%). Surprisingly,the presence of water (6.2%) did not give rise to adetectable water evaporation endothermic peakon the differential scanning calorimetry curve.

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–68 61

Fig. 1. Normalized differential scanning calorimetry curves for econazole b-cyclodextrin complex, molar ratio 2:3, and thecorresponding physical mixture. Econazole melting point 87°C.

The cut-off temperature in Fig. 1 is 110°C. Othercurves were run to 160°C without endothermic orexothermic peaks being present.

According to Szejtli (1988), evaporation of thewater content from a cyclodextrin inclusion com-plex will often take place below 100°C whereasevaporation of crystal water from neat b-cy-clodextrin is seen just above 100°C. The differencein evaporation temperature is caused by differentstrengths of the bonds between water moleculesand cyclodextrin molecules. The reason why waterevaporation was not detectable in the present casecould be that the evaporation took place over abroad temperature interval or the water moleculeswere bound so efficiently that evaporation did nottake place at all.

Three batches of complex were analyzed foreconazole, b-cyclodextrin and water. The com-plex composition was econazole 17.090.3%, b-cyclodextrin 7693% and water 6.290,17%;n=3, 9S.D. That is, the molar ratio was(2:3:15.5). An econazole b-cyclodextrin inclusioncomplex with a 1:1 molar ratio of econazole andb-cyclodextrin has been reported previously(Pedersen et al., 1993a). The ratio determinationwas based solely on an econazole analysis, i.e.analysis of b-cyclodextrin and water was not car-ried out.

In the present study, the isolation conditions,i.e. temperature and pH were varied in the ranges

20–26°C and 6.0–10.7 to see if the differencebetween the present and previously reported mo-lar ratio was due to a variation of the isolationconditions. It did not seem to be the case. Themolar ratio econazole:b-cyclodextrin was 2:3 andit did not vary with temperature and pH.

X-ray powder diffraction analysis results forthe two complexes, molar ratio 1:1 and 2:3 aredepicted in Fig. 2. Minor differences between thetwo X-ray diffraction patterns are visible.

As a part of the biological experiments physicalmixtures of econazole and b-cyclodextrin wereprepared by gentle mixing in mortars. Differentialscanning calorimetry curves of physical mixturesamples mixed for various times, 1–10 min,showed that the area of the endothermic peakcorresponding to the melting of econazole de-creased with increasing mixing time. A physicalmixture of econazole and b-cyclodextrin was pre-pared by vigorous mixing in a mortar for 10 min.A small amount of water was added during themixing to make the product paste like. The finalproduct composition, after drying at 60°C for 3days, was 16.0% econazole, 74–80% b-cyclodex-trin and 7.0% water. An econazole melting peakwas not present on the differential scanning curve(data not shown). Due to the risk of changingeconazole’s properties, the physical mixtures ap-plied in the present studies were prepared bygentle mixing in a mortar for 1 min.

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–6862

Fig. 2. X-ray diffraction patterns: (1) b-cyclodextrin; (2) econazole; (3) econazole:b-cyclodextrin, molar ratio 1:1; and (4)econazole:b-cyclodextrin, molar ratio 2:3. Exposure time 1 h.

3.2. Antimycotic acti6ity in a fluid medium

The effect of the econazole b-cyclodextrin com-plexes, molar ratio 1:1 and 2:3 upon Candidaalbicans, PF 1383 88, in vitro is depicted in Fig. 3.The effect of econazole, econazole nitrate andphysical mixtures of econazole/econazole nitrateand b-cyclodextrin, molar ratio 1:1 and 2:3 isincluded too. The genuine complexes were muchmore active than the physical mixtures, econazolealone and econazole nitrate alone. The 2:3 com-plex was the more active of the two complexes.After 2-h exposure to the complexes, the survivingCandida albicans did not recover and makecolonies within a 48-h incubation period. After96-h incubation colonies were present. Regardingthe other treatments, i.e. physical mixtures, econa-zole and econazole nitrate, the surviving Candidaalbicans organisms recovered and grew to visiblecolonies within a 48-h incubation period (Fig. 3a).After 8- and 24-h exposure of the Candida albi-cans, the recovery rate for the different treatmentswas more equal (Fig. 3b and c).

3.3. Dissolution rate and supersaturation

The solubility diagram for econazole nitrateand b-cyclodextrin in the microbiological growthmedium at 3791°C is depicted in Fig. 6. Thediagram might be of the Ap type. All over theb-cyclodextrin concentration range, the solidphase in the tubes was econazole base. The iden-tity of the solid phase was confirmed by HPLCand differential scanning calorimetry (data notshown). Regarding the diagram type, an alterna-tive interpretation of the solubility diagram, Fig.6, may be that proteins and other components

from the growth medium competed with econa-zole for the cyclodextrin cavities. Up to 4 mg/mlb-cyclodextrin, almost no cavities were availablefor econazole. Above 4 mg/ml some of cavitieswere available for econazole. If this is the case,the diagram is of the Al type above 4 mg/mlb-cyclodextrin.

The results of the dissolution tests — per-formed in microbiological growth medium, pH7.5, 3791°C — for econazole, econazole nitrate,the physical mixtures of econazole or econazolenitrate and b-cyclodextrin and the inclusion com-plex 2:3 are depicted in Fig. 4. Throughout thedissolution studies it was ensured by measurementthat the pH was kept at 7.5. Due to the extremelylow solubility of econazole and econazole nitrate,the concentration of antimycotic during the disso-lution rate testing of the drugs alone was hardlydetectable, B0.5 mg/ml. On the contrary, thephysical mixture of econazole nitrate and b-cy-clodextrin gave rise to a fast drug dissolution rate.This dissolution rate curve is typical for a systemcausing drug supersaturation of the dissolutionmedium (Szejtli, 1988). Probably, the supersatura-tion was caused by conversion of the nitrate saltto econazole base. The drug supersaturationmight be the reason why the econazole nitrateb-cyclodextrin physical mixture had a quite sig-nificant effect upon the Candida albicans (Fig. 3).

Regarding the inclusion complex and the physi-cal mixture, both with the molar compositioneconazole:b-cyclodextrin 2:3, the drug dissolutionrate from the physical mixture was faster than thedissolution rate from the inclusion complex (Fig.4). The results indicate that the solubility of theeconazole b-cyclodextrin complex in the mediumwas quite low. Neither the physical mixture nor

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–68 63

Fig. 3. Antimycotic effect of various econazole compositions upon Candida albicans. comp, inclusion complex of econazole andb-cyclodextrin; phy, physical mixture of econazole (nitrate) and b-cyclodextrin; 1:1 and 2:3, molar ratio of econazole andb-cyclodextrin.

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–6864

Fig. 4. , econazole b-cyclodextrin solubility diagram in microbiological growth medium at 37°C. Corresponding b-cyclodextrinand econazole concentrations during dissolution rate testing in the medium. �, econazole b-cyclodextrin inclusion complex, molarratio 2:3; �, econazole b-cyclodextrin physical mixture, molar ratio 2:3. An amount corresponding to 3.3 mg econazole per ml wasadded.

the inclusion complex gave a dissolution ratecurve indicating that a drug supersaturation ofthe dissolution medium was present (Fig. 4). Thatis, the results of the dissolution rate study did notcorrelate with the microbiological results (Fig. 3).The antimycotic effect of the 2:3 inclusion com-plex was much more pronounced than the effectof the econazole b-cyclodextrin 2:3 physical mix-ture. Although the econazole nitrate b-cyclodex-trin physical mixture gave rise to an extremely fasteconazole dissolution rate and drug supersatura-tion, the antimycotic effect was not as pro-nounced as the effect of the inclusion complex.

Van Doorne et al. (1988) studied the influenceof b-cyclodextrin upon the antimycotic activity ofimidazole derivatives such as econazole. Complex-ation between antimycotic and b-cyclodextrin in asemi-solid medium decreased the antimycotic ac-tivity. Van Doorne et al. concluded that the dis-solved antimycotic b-cyclodextrin complex onlypossessed a minor antimycotic activity if any.Recently, part inhibition of the antimycotic effectof dissolved miconazole and econazole by com-plexation with b-cyclodextrin was reported(Pedersen et al., 1993b). The previous studiesindicate that the high antifungal activity of the

present econazole b-cyclodextrin inclusion com-plexes hardly could be due to a high antimycoticeffect of the complexes in their own right.

Based on the above, it is difficult to understandwhy the inclusion complex had higher antimycoticactivity than the physical mixture of econazoleand b-cyclodextrin. The reason for the lack ofcorrelation between antimycotic effect and disso-lution rate/supersaturation may be that tendencyof the inclusion complex to supersaturate thegrowth medium was underestimated by the tradi-tional way of disclosing drug supersaturation. Thepossible underestimation of the drug supersatura-tion could be due to the fact that the b-cyclodex-trin concentration in the growth medium is notconsidered by the traditional method. The disso-lution rate for b-cyclodextrin from the physicalmixture and the inclusion complex of econazoleand b-cyclodextrin, both with a molar ratio 2:3, isdepicted in Fig. 5. The cyclodextrin dissolutionrate was higher from the physical mixture thanfrom the econazole b-cyclodextrin complex. Dueto the complexation between econazole and b-cy-clodextrin in the growth (dissolution) medium(Fig. 4), considering the b-cyclodextrin concentra-tion may be of importance when the drug super-

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–68 65

Fig. 5. b-cyclodextrin dissolution rate in microbiological growth medium, 37°C, average and S.E.M., n=3. An amountcorresponding to 3.3 mg econazole per ml was added. �, econazole:b-cyclodextrin inclusion complex, molar ratio 2:3; �,econazole:b-cyclodextrin physical mixture, molar ratio 2:3.

saturation is estimated. The solubility diagram andthe paired or corresponding concentrations ofeconazole and b-cyclodextrin from the dissolutionrate studies (Figs. 4 and 5), are plotted in Fig. 6.Regarding the dissolution rate study of the physicalmixture, all the points are placed below the solubil-

ity curve, whereas some of the points from theinclusion complex study are placed above or at thesolubility curve. Other points are situated below thecurve (Fig. 6). Points situated below the solubilitycurve correspond to the dissolution medium beingunsaturated with econazole, while points placedabove the curve correspond to the medium beingsupersaturated. Because the points are comparedwith the solubility curve, the complexation betweeneconazole and b-cyclodextrin is taken into consid-eration when the drug supersaturation of thegrowth medium is evaluated by this way. Accordingto Fig. 6, the inclusion complex gave rise to drugsupersaturation whereas the physical mixture didnot. In fact, the physical mixture was far away fromjust saturating the dissolution medium with econa-zole. That is, regarding drug supersaturation thepicture presented in Fig. 6 is controversial whencompared to the one in Fig. 4. The reason for thecontroversy is that the traditional method of dis-closing supersaturation (Fig. 4) does not take intoaccount the concentration of cyclodextrin in thedissolution medium and the complexation betweendrug and cyclodextrin.

The reason why the econazole b-cyclodextrininclusion complex worked better than the physicalmixture with respect to antimycotic effect is likelyto be due to the supersaturation phenomenondisclosed by the method illustrated in Fig. 6.

Fig. 6. , econazole b-cyclodextrin solubility diagram inmicrobiological growth medium at 37°C. Corresponding b-cy-clodextrin and econazole concentrations during dissolutionrate testing in the medium. �, econazole b-cyclodextrin inclu-sion complex, molar ratio 2:3; �, econazole b-cyclodextrinphysical mixture, molar ratio 2:3. An amount correspondingto 3.3 mg econazole per ml was added.

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–6866

Fig. 7. Normalized differential scanning calorimetry curve of precipitate after 24-h dissolution rate testing of econazole b-cyclodex-trin inclusion complex, molar ratio econazole:b-cyclodextrin 2:3. Econazole melting point 87°C.

In each dissolution rate experiment, econazoleor econazole nitrate corresponding to 3.3 mg/mleconazole base was applied. For the 2:3 econa-zole:b-cyclodextrin physical mixture and complex3.3 mg/ml econazole corresponded to 14.8 mg/mlb-cyclodextrin. According to Fig. 5, all the b-cy-clodextrin from the physical mixture went intosolution within 20 min, while, surprisingly, only 8mg/ml of the cyclodextrin in the inclusion com-plex were dissolved after 24 h. Regarding theinclusion complex, the precipitate in two of thedissolution rate tubes was isolated after 24 h andthe econazole content was measured by HPLC.The results were an econazole content of 27.1 and28.9%, respectively. Taking into consideration theconcentration of b-cyclodextrin in the 15 ml dis-solution medium after 24 h, i.e. 8 mg/ml, theamount of inclusion complex added initially 289.5mg, the percentage of econazole and water in theinclusion complex 17.0% and 6.2% respectively,

and finally the percentage of econazole in theprecipitate after 24-h dissolution experiment28.0%, the precipitate consisted of 20 mg econa-zole and 134 mg inclusion complex with the molarcomposition econazole:b-cyclodextrin:water2:3:15.5. That is, even after 24 h only about onehalf of the inclusion complex added initially wastransformed to free econazole and b-cyclodextrin.

The presence of a neat econazole part in theprecipitate after 24 h was detectable by differen-tial scanning calorimetry (Fig. 7).

Applying the method illustrated in Fig. 6 maybe worthwhile when studying other cyclodextrininclusion complexes.

3.4. Hemolysis

The effect of b-cyclodextrin on the hemolysis isdepicted in Fig. 8. The dose response study was inagreement with results previously reported by

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–68 67

Fig. 8. Hemolysis (%) as a function of b-cyclodextrin concen-tration. Incubation time 15 and 30 min.

Fig. 10. TEER (V cm2) of TR146 cell layers during exposureto 20.2 mg/ml Comp/Phy 2:3 (econazole b-cyclodextrin com-plex/physical mixture, molar ratio 2:3).

Ohtani et al. (1989). According to Fig. 9, theeconazole b-cyclodextrin complexes, 1:1 or 2:3,caused significant hemolysis. The physical mix-tures were considerably less hemolytic than thecomplexes. Probably, the higher hemolytic activ-

ity of the complexes was due to an improvedavailability of the antimycotic drug. Higheravailability of econazole may also be the reasonwhy econazole nitrate caused more hemolysisthan the econazole base (Fig. 9). The lower b-cy-clodextrin dissolution rate from the 2:3 inclusioncomplex compared with the corresponding physi-cal mixture (Fig. 5) should decrease the hemolyticactivity caused by the cyclodextrin part of theinclusion complex.

3.5. TR146 cell culture

Measurement of TEER is a way to evaluate theintegrity of TR146 cell layers grown on filters(Jacobsen et al., 1995).

According to Fig. 10, neither the physical mix-ture nor the inclusion complex, both with themolar ratio econazole:b-cyclodextrin 2:3, affectedthe TEER value within the 4-h test period, al-though 20.2 mg/ml physical mixture or inclusioncomplex was applied on the apical side of the celllayer. The antimycotic alone 3.3 mg/ml, b-cy-clodextrin alone 16.9 mg/ml or pure Hank’sbuffer did not affect the TEER value either (datanot shown).

Fig. 9. Hemolysis (%) of various econazole compositions.comp, inclusion complex of econazole and b-cyclodextrin;phy, physical mixture of econazole (nitrate) and b-cyclodex-trin; 1:1 and 2:3, molar ratio of econazole and b-cyclodextrin.For cyclodextrin containing samples, an amount correspond-ing to 7.8 mg b-cyclodextrin per ml was added; 1.6 and 1.9 mgper ml were added of econazole and econazole nitrate, respec-tively.

M. Pedersen et al. / International Journal of Pharmaceutics 165 (1998) 57–6868

Fig. 11. Mortality of TR146 cells after 240-min exposure to20.2 mg/ml Comp/Phy 2:3 (econazole b-cyclodextrin complex/physical mixture, molar ratio 2:3), 3.3 mg/ml econazole and3.9 mg/ml econazole nitrate.

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An analysis of the protein concentration in thebuffer after TR146 cells were exposed to eitherinclusion complex, physical mixture, econazole,econazole nitrate or pure Hank’s buffer for 30 or240 min did not reveal a significant differenceregarding the influence of the various treatmentsupon protein liberation or extraction from thecells (data not shown). Trypan blue determinationof the TR146 cell viability after 240-min exposureindicated that the physical mixture and econazolenitrate were more toxic to TR146 than the econa-zole base and surprisingly also the inclusion com-plex (Fig. 11).

Acknowledgements

This research was supported by grants fromGeneralkonsul Ludvig Tegner og hustrus Mindel-egat. Birgit Bellers is acknowledged for linguisticreviewing of this paper.

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