A genuine clotrimazole γ-cyclodextrin inclusion complex-isolation, antimycotic activity, toxicity...

International Journal of Pharmaceutics 176 (1998) 121–131

A genuine clotrimazole g-cyclodextrin inclusioncomplex-isolation, antimycotic activity, toxicity and an unusual

dissolution rate

Morten Pedersen a,*, Simon Bjerregaard b, Jette Jacobsen 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 7 September 1998; accepted 24 September 1998

Abstract

A crystalline clotrimazole g-cyclodextrin inclusion complex, molar ratio 1:1.0, was isolated from phosphate buffer0.05 M, pH 7.1. Due to the low water solubility of the inclusion complex, the clotrimazole dissolution rate from thecomplex was very low. However, application of a new method to disclose supersaturation phenomena showed that thecomplex gave rise to a profound clotrimazole supersaturation during the dissolution rate test. Probably, theclotrimazole supersaturation was the reason why the inclusion complex had higher antimycotic activity than bothclotrimazole and a physical mixture of clotrimazole and g-cyclodextrin, molar ratio 1:1.0. In addition, the inclusioncomplex was the most toxic on human erythrocytes and on non-differentiated monolayers of epithelial like humanTR146 cells. On the other hand, no difference in the toxicity of the inclusion complex, the physical mixture andclotrimazole was observable when robust multilayers of differentiated TR146 cells were exposed to the compounds.© 1998 Published by Elsevier Science B.V. All rights reserved.

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

1. Introduction

Cyclodextrins are oligosaccharides which areable to form complexes with lipophilic drugs orlipophilic parts of drugs, thus changing their* Corresponding author. Tel.: +45 97821321.

0378-5173/98/$ - see front matter © 1998 Published by Elsevier Science B.V. All rights reserved.

PII S0378-5173(98)00310-X

M. Pedersen et al. / International Journal of Pharmaceutics 176 (1998) 121–131122

physicochemical and biopharmaceutical propertiesin a desirable manner.

Isolation of genuine b-cyclodextrin complexes ofthe antimycotic lipophilic drugs, econazole andmiconazole has been reported previously (Pedersenet al., 1993a, 1998; Pedersen, 1994). Other studiesnot resulting in isolation of genuine complexes ofcyclodextrins and imidazole antimycotics were car-ried out by Piel et al. (1997, 1998), Tenjarla et al.(1998), Okimoto et al. (1996), Esclusa-Diaz et al.(1996a,b), Gerloczy et al. (1995), Pedersen (1993),Pedersen et al. (1993b), Mura et al. (1992), Pearsonand Salole (1989), Bononi et al. (1988) and VanDoorne et al. (1988a,b).

The main aim of the present study was to isolatea new genuine cyclodextrin inclusion complex ofthe antimycotic clotrimazole, and to study theantimycotic, toxicological, and physicochemicalproperties of the isolated inclusion complex. Thetoxicology of the complex was tested on humanerythrocytes and on a newly established humanbuccal cell culture model based on the cell lineTR146 (Jacobsen et al., 1995, 1996). Regarding thephysicochemical properties, a recently publishedmethod was included to disclose possible clotrima-zole supersaturation episodes during the dissolu-tion rate testing of the cyclodextrin inclusioncomplex (Pedersen, 1997).

2. Experimental

2.1. Materials

Clotrimazole and b-cyclodextrin were purchasedfrom Sigma (St. Louis, MO, USA) and g-cyclodex-trin was purchased from Cyclolab (Hungary). a-Cyclodextrin was from AVEBE (The Netherlands).Candida albicans PF 1383 88 was generously sup-plied by Statens Seruminstitut (Copenhagen, Den-mark). Dulbecco’s modified Eagle medium(DMEM; Cat. No. B-1001) was purchased fromHyClone® Laboratories (Logan, UT, USA), andpenicillin (104 units/ml)/streptomycin (104 mg/ml)solution was obtained from Gibco BRL (Paisley,UK). Heat-inactivated fetal calf serum was pur-chased from Sera-Lab (Sussex, UK). The continouscell line TR146, derived from a human neck mode

metastasis originating from a buccal carcinoma(Rupniak et al., 1985), was kindly provided byImperial Cancer Research Technology (London,UK). Falcon cell culture inserts (polyethyleneterephthalate, 1.6×106 pores/cm2, pore diameter0.45 mm, growth area 4.2 cm2) and Falcon 6-wellculture plates (tissue culture treated polystyrene)were from Becton Dickinson Labware (New Jersey,NJ, USA). Nunclon™ Delta Surface MicrowellPlates 96-well flat bottomed (polystyrene, certifiedsurface treatment) was obtained from NNI NuncA/S (Roskilde, Denmark).

All other chemicals were of analytical grade.

2.2. Method

2.2.1. Solubility diagrams and isolation ofinclusion complex

Solubility measurements were carried out asdescribed by Higuchi and Connors (1965). In 5 mlof distilled water up to 18 mg/ml b-cyclodextrinwas dissolved and 10 mg clotrimazole was sus-pended. Similarly, the solubility diagram was deter-mined for clotrimazole and b-cyclodextrin inammonium phosphate buffer, 0.05 M, pH 7.1 or10.0, and at various temperatures, 6 and 21°C.

Solubility diagrams of clotrimazole and a-cy-clodextrin up to 120 mg/ml, and g-cyclodextrin upto 225 mg/ml were measured at 21°C in 0.05 Mdiammonium phosphate buffer, pH 7.1. As above,10 mg clotrimazole and 5 ml buffer were applied.

Throughout the solubility studies, the sampleswere rotated at least 10 days for equilibration totake place. Afterwards, the samples were filteredthrough 0.2-mm Sartorius cellulose acetate mem-brane filters. The concentration of antimycotic inthe filtrate was analysed by a HPLC method.

A solid clotrimazole g-cyclodextrin inclusioncomplex was isolated at 21°C by shaking a 0.5 lscrew cap bottle containing 0.5 l diammoniumphosphate buffer 0.05 M, pH 7.1, in which 2 mg/mlclotrimazole was suspended and 60 mg/ml g-cy-clodextrin was dissolved initially. The isolationconditions corresponded to a point on the descend-ing part of the Bs solubility diagram.

During the shaking period, homogeneuos sam-ples were taken from the bottle and dried. The driedproducts were analysed by differential scan-

M. Pedersen et al. / International Journal of Pharmaceutics 176 (1998) 121–131 123

ning calorimetry to measure the residue of crys-talline clotrimazole. After ten days of shaking,there was no longer crystalline clotrimazole in thedried product, i.e. the termodynamic equilibriumhad been reached, and all clotrimazole had beenconverted to the solid clotrimazole g-cycloddex-trin complex, except for the insignificantlyamount of clotrimazole dissolved in the solubilitymedium. After the equilibrium was reached, thesolid complex was isolated by paper filtration.The complex was washed with a few millilitres ofwater and dried at 60°C for 3 days. The driedproduct was analysed for the content of clotrima-zole, g-cyclodextrin, and water.

A physical mixture of clotrimazole and g-cy-clodextrin, molar ratio 1:1.0, was prepared bygently mixing in a mortar.

2.2.2. 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 aluminium 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.

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

recorded with a Guinier, XDC 700 IRDAB pow-der diffraction camera using a Philips PW 1720X-ray generator. Cr Ka radiation was applied.

2.2.4. HPLC methodsThe concentration of clotrimazole was mea-

sured by a previously published reversed phaseHPLC method (Pedersen, 1993). The dried (3days at 60°C) clotrimazole g-cyclodextrin inclu-sion complex was dissolved in dimethyl for-mamide before the clotrimazole analysis. Theg-cyclodextrin content of the dried complex wasmeasured by the g-cyclodextrin HPLC reversedphase method described below. Before the analy-sis the complex was dissolved in dimethyl for-mamide. The g-cyclodextrin concentration in thegrowth medium/dissolution medium was mea-sured by the same reversed phase HPLC method.The eluent was composed of 7% (v/v) methanol

and 93% (v/v) deionised water, the column was aMerck Lichrosorb 100 RP18 (4×125 mm)equipped with a Lichrosorb 100 RP18 guardcolumn. A Merck refractive index detector wasapplied. The quantitation limit for a 20-m l loopwas 0.5 mg/ml g-cyclodextrin. The correlationcoefficient was 0.999 for the concentration range0.5–6 mg/ml.

2.2.5. Determination of water contentThe water content of the dried (3 days at 60°C)

clotrimazole g-cyclodextrin inclusion complex wasmeasured by a Karl Fischer titration procedure,previously described (Pedersen et al., 1998).

2.2.6. Antimycotic acti6ityCultures of Candida albicans were grown for

2.5 h at 3791°C in 10 ml samples of sterileglucose, peptone, yeast extract growth medium(Pedersen and Rassing, 1990), which was adjustedto pH 7.5 with sterile 0.5 M disodium hydrogenphosphate buffer. The initial number of yeast cellswas 5×105 per ml. After the 2.5 h growth period,clotrimazole 1.5 mg/ml, g-cyclodextrin 5.7 mg/ml,clotrimazole g-cyclodextrin inclusion complex7.65 mg/ml, molar ratio 1:1.0 or clotrimazoleg-cyclodextrin physical mixture 7.65 mg/ml, molarratio 1:1.0 were added. The compounds weresieved through a 300-mm sieve just before theaddition. Samples of 100 m l were taken from thegrowth medium tubes during a 24 h experimentalperiod. The samples were diluted appropriatelyand the dilutions were plated on agar plates. Afterincubation of the agar plates for 192 h at 33°C,colony counting was carried out.

2.2.7. Dissolution rate studies in Candida growthmedium

The dissolution rate studies of clotrimazole 1.5mg/ml, dried clotrimazole g-cyclodextrin inclusioncomplex 7.65 mg/ml and physical mixture ofclotrimazole and g-cyclodextrin 7.65 mg/ml werecarried out using the same experimental condi-tions as during the testing of the antimycoticactivity. Samples were taking from the tubes andimmediately filtered through Sartorius celluloseacetate 0.2-mm filters, and the concentration ofclotrimazole and g-cyclodextrin in the filtrates


was measured immediately by HPLC methods.Regarding the inclusion complex, the solid phasein one of the tubes after 24 h testing was isolatedby paper filtration. The solid phase was washedwith a few drops of water, dried at 60°C for 3days and analysed for the clotrimazole content.

2.2.8. HemolysisErythrocytes were separated by centrifugation

of citrated human blood, washed three times withisotonic phosphate buffer and resuspended in thebuffer to give a hematocrit value of 5% (Ohtani etal., 1989). Buffer (3.8 ml) and 0.2 ml erythrocytesuspension (5%) were mixed. Test substances, i.e.clotrimazole 3.1 mg/ml, g-cyclodextrin 12.0 mg/ml, clotrimazole g-cyclodextrin inclusion complex16 mg/ml and physical mixture of clotrimazoleand g-cyclodextrin 16 mg/ml were added as solidcompounds at time zero. The samples were incu-bated in a 37°C water bath for 15 min. After-wards, the samples were centrifuged for 3 min at2000×g. The absorbance of the supernatant wasmeasured at 543 nm. A 100% hemolysis ab-sorbance value was obtained by mixing 0.2 mlerythrocyte suspension and 3.8 ml distilled waterinstead of isotonic buffer, and incubating it for 15min at 37°C and measuring the absorbance at 543nm (Ohtani et al., 1989).

2.2.9. Toxicity on TR146 cell cultureTR146 cells were incubated and maintained in

75-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 heat-inactivated fetalcalf serum 10%, glucose 3.5 mg/ml and penicillin100 units/ml/streptomycin 100 mg/ml. Further de-tails concerning maintenance and seeding ofTR146 cells were as described previously (Jacob-sen et al., 1995). Regarding seeding and growth ofTR146 cells on Falcon filters, the procedure de-scribed previously (Pedersen et al., 1998) wasfollowed strictly. After 29 days’ growth on thefilters, the cell layers were applied for transepithe-lial electrical resistance measurements. The effecton the transepithelial electrical resistance of ex-posing the cell layers to clotrimazole 3.1 mg/ml,g-cyclodextrin 12 mg/ml, clotrimazole g-cyclodex-

trin inclusion complex 16 mg/ml and a physicalmixture of clotrimazole and g-cyclodextrin 16 mg/ml was estimated during a 4 h exposure period.The transepithelial electrical resistance measure-ment on TR146 cell layers was carried out asdescribed by Pedersen et al. (1998).

Immediately after the 4 h period, the cell layerswere removed by trypsination, and 500 m l cellsuspension was mixed with 500 m l 0.4% TrypanBlue Solution (Sigma catalogue 1530-1531, 1992).The TR146 viability was calculated by countingdead cells (stained blue) and total number of cellsusing an inverted light microscope and a graticule.

A MTS/PMS toxicity assay on TR146 cellmonolayers, grown for 24 h in a 96-well plate,was carried out as described by Jacobsen et al.(1996). The MTS/PMS assay is a tetrazoliumsalt-based colorimetric assay determining the cel-lular viability by means of cellular dehydrogenaseactivity and hence reduction of the tetrazoliumsalt, MTS, to the corresponding coloured MTS-formazan. Presence of the electron couplingreagent, PMS, speeds up the formation of MTS-formazan (Jacobsen et al., 1996).

The MTS/PMS toxicity of clotrimazole 0.19,0.78, and 3.1 mg/ml, g-cyclodextrin 0.75, 3.0, and12 mg/ml, clotrimazole g-cyclodextrin inclusioncomplex 1, 4, and 16 mg/ml and finally clotrima-zole g-cyclodextrin physical mixture 1, 4, and 16mg/ml was measured. Sodium dodecylsulfate10−2 M was used as a positive control of theMTS/PMS toxicity procedure.

3. Results and discussion

3.1. Solubility diagrams

Van Doorne et al. (1988b) reported that clotri-mazole gave an An solubility diagram with a-cy-clodextrin in distilled water at room temperature.In the present study of a-cyclodextrin and clotri-mazole the solubility diagram in phosphate buffer0.05 M, pH 7.1 at room temperature was of theAp type, i.e. no sedimentation of a-cyclodextrininclusion complex took place (data not shown).Previously, it was shown that application of 0.05M phosphate buffer instead of water caused an


econazole nitrate b-cyclodextrin solubility dia-gram to switch from the An to the Bs type(Pedersen et al., 1993a).

The solubility diagrams for clotrimazole andb-cyclodextrin in different media are depicted inFig. 1. Each solubility diagram in Fig. 1 is basedon one determination. According to the figure,distilled water at 21°C gave a Bs diagram. How-ever, the b-cyclodextrin concentration at whichformation of solid inclusion complex started wasquite near b-cyclodextrin’s own solubility. Theformed inclusion complex was of a colloid likenature and it was not possible to isolate thecomplex by filtration or centrifugation. Applica-tion of 0.05 M phosphate buffer pH 7.1 instead ofwater resulted in an A type solubility diagram. Atype solubility diagrams were also the result whendistilled water at 6°C, Fig. 1 or phosphate buffer0.05 M, pH 10 was applied as medium (data notshown). Lowering the temperature a few degreesor increasing the pH value from 7.1 to 10.0 werepreviously applied to get a Bs solubility diagramfor b-cyclodextrin and the imidazole derivativemiconazole (Pedersen, 1994). However, it did notwork for clotrimazole and b-cyclodextrin.

g-Cyclodextrin gave a Bs type solubility dia-gram with clotrimazole, Fig. 2. According to theequation,

K1,1=slope/So(1−slope)

where So is the intrinsic solubility of clotrimazole0.61×10−6 M (Pedersen, 1993), and the initial

Fig. 2. Clotrimazole g-cyclodextrin solubility diagram at 21°Cin phosphate buffer 0.05 M, pH 7.1.

slope of the solubility diagram in Fig. 2 is 6.62×10−4,

K1,1=1086 M−1.

The DSC results of the clotrimazole g-cyclodex-trin complex, a physical mixture of drug andg-cyclodextrin, same ratio as for the complex (seebelow), clotrimazole and g-cyclodextrin indicatedthat the isolated complex did not contain aresidue of clotrimazole, Fig. 3. No clotrimazolemelting peak was present during the DSC analysisof the inclusion complex. The content of clotrima-zole, g-cyclodextrin and water in the isolated,washed, and dried complex was 19.790.2 (n=3,9S.E.M.), 77.791.2 (n=3, 9S.E.M.) and5.690.2% (n=3, 9S.E.M), respectively. The re-sults are the average of the content of threebatches. The molar ratio of the complex wasclotrimazole:g-cyclodextrin:water, 1:1.0:5.4. Ac-cording to the X-ray diffraction patterns, the in-clusion complex was crystalline and the patternfor the complex differed from the patterns of thephysical mixture, clotrimazole and g-cyclodextrin,Fig. 4.

3.2. Antimycotic acti6ity

The antimycotic effect of clotrimazole 1.5 mg/ml, physical mixture of clotrimazole and g-cy-clodextrin 7.65–1.5 mg/ml clotrimazole and 5.7mg/ml g-cyclodextrin, the inclusion complex7.65–1.5 mg/ml clotrimazole and 5.7 mg/ml g-cy-clodextrin, and g-cyclodextrin 5.7 mg/ml is de-picted in Fig. 5. The inclusion complex was by far

Fig. 1. Clotrimazole b-cyclodextrin solubility diagrams indifferent media and at different temperatures. pH in buffer 7.1.


Fig. 3. Normalized DSC curves for (1) clotrimazole; (2) physical mixture of clotrimazole and g-cyclodextrin, molar ratio 1:1.0; (3)g-cyclodextrin; and (4) clotrimazole g-cyclodextrin inclusion complex, molar ratio 1:1.0.

the most active of the tested compounds. Thephysical mixture and clotrimazole alone hadabout the same antimycotic activity, while g-cy-clodextrin was practically without antimycoticeffect.

A comparison of the antimycotic effect, theclotrimazole dissolution rate and the clotrimazolesupersaturation tendency indicated that the clotri-mazole supersaturation, which is disclosed in Fig.8, was of significant importance for the superiorantimycotic activity of the inclusion complex (seeSection 3.3).

3.3. Dissolution rate studies in Candida growthmedium

The clotrimazole dissolution rate from neat

clotrimazole 1.5 mg/ml, the physical mixture ofclotrimazole and g-cyclodextrin and the inclusioncomplex both with the molar ratio 1:1.0, is depictedin Fig. 6. The dissolution medium was the Candidaalbicans growth medium and 7.65 mg/ml wasapplied of the complex and the physical mixture,corresponding to 1.5 mg/ml clotrimazole. Theinitial dissolution rate for the inclusion complexand the physical mixture, i.e. the rate within thefirst 5 min was about the same for the twocompounds. Both had a faster dissolution rate thanneat clotrimazole. Apparently, equilibrium wasestablished for the physical mixture already 5 minafter applying it to the dissolution medium.Regarding the inclusion complex, the dissolutionrate curve was more complicated. The concentra-


Fig. 4. X-ray diffraction patterns: (1) clotrimazole; (2) g-cyclodextrin; (3) physical mixture of clotrimazole and g-cyclodextrin, molarratio 1:1.0; and (4) clotrimazole g-cyclodextrin inclusion complex, molar ratio 1:1.0. (5) Precipitate isolated after 24 h. Dissolutionrate experiment with the clotrimazole g-cyclodextrin inclusion complex. Exposure time 1 h.

tion of dissolved clotrimazole reached its maxi-mum after 5 min. Afterwards the clotrimazoleconcentration dropped throughout the rest of thetest period (24 h). The drop in the concentrationmight indicate an initial clotrimazole supersatura-tion. However, it is unusual that the clotrimazoleconcentration during the complex dissolution test-ing dropped so much below the clotrimazole con-centration which was obtained during the testingof the physical mixture. Fig. 7 shows the g-cy-clodextrin concentrations during the dissolutionrate testing of the physical mixture and the inclu-sion complex. All the g-cyclodextrin contained inthe physical mixture was dissolved within 5 min,whereas the g-cyclodextrin dissolution rate fromthe inclusion complex was much slower. Only 1.1mg/ml g-cyclodextrin was dissolved after 24 h.The initial amount of added inclusion complex7.65 mg/ml contained 5.7 mg/ml g-cyclodextrin.Analysis of the clotrimazole, g-cyclodextrin andwater content of the washed and dried cyclodex-trin inclusion complex was actually carried out(see Section 3.1). That is the low g-cyclodextrindissolution rate from the inclusion complex wasnot due to a lower cyclodextrin content than

expected. The slow dissolution rate for the inclu-sion complex was not due to a Bs solubilitydiagram for clotrimazole and g-cyclodextrin inthe dissolution medium. According to Fig. 8, thediagram was of the A type, at least up to 6 mg/ml.That is, the dissolution of the inclusion complexwas not hindered by the complex becoming thestable solid phase during the dissolution ratestudy.

The initial slope of the solubility diagram ofclotrimazole and g-cyclodextrin in the growthmedium (Fig. 8) was 0.0144 and clotrimazole’ssolubility in the medium wasB0.5 mg/ml–1.5×10−6 M. The stability constant K1,1 was\10076M−1, according to the K1,1 equation shown previ-ously. The stability constant in growth medium,pH 7.5, was about a factor of ten higher than thestability constant in 0.05 M phosphate buffer, pH7.1. The higher pH value in the growth mediumwill probably increase the stability constant be-cause clotrimazole is neutralized at higher pH,pKa in 50% aqueous ethanol was reported to be4.7 (Buechel et al., 1972). However, a more im-portant reason to the increased stability constantin the growth medium may be, that the medium


Fig. 5. Antimycotic effect of various compositions upon Candida albicans. �, blank; ", 5.7 mg/ml g-cyclodextrin; , 7.65 mg/mlphysical mixture clotrimazole g-cyclodextrin, molar ratio 1:1.0; + , clotrimazole 1.5 mg/ml; , inclusion complex clotrimazoleg-cyclodextrin, molar ratio 1:1.0.

contained 4% w/w of the water structure formingagent glucose. Other water structure formingagents, i.e. sorbitol and fructose, were previouslyshown to increase the stability constant betweenclotrimazole and b-cyclodextrin (Pedersen, 1993).

Comparison of the solubility diagram and thepaired or corresponding concentrations of clotri-mazole and g-cyclodextrin during the dissolution

rate studies was done in Fig. 8 (Pedersen, 1997).According to the figure, the inclusion complexgave rise to significant clotrimazole supersatura-tion of the dissolution medium, whereas supersat-uration did not take place when the physicalmixture was tested. Supersaturation is present ifthe paired concentration points are placed abovethe solubility diagram curve (Pedersen, 1997). Theclotrimazole supersaturation that the inclusion

Fig. 6. Clotrimazole dissolution rate in microbiological growthmedium, 37°C, average and S.E.M., n=3. An amount corre-sponding to 1.5 mg/ml clotrimazole was added. , 7.65 mg/mlphysical mixture clotrimazole g-cyclodextrin, molar ratio 1:1.0;, 7.65 mg/ml inclusion complex clotrimazole g-cyclodextrin,molar ratio 1:1.0; + , 1.5 mg/ml clotrimazole.

Fig. 7. g-Cyclodextrin dissolution rate in microbiologicalgrowth medium, 37°C, average and S.E.M., n=3. An amountcorresponding to 1.5 mg/ml clotrimazole was added. , 7.65mg/ml physical mixture clotrimazole g-cyclodextrin, molarratio 1:1.0; , 7.65 mg/ml inclusion complex clotrimazoleg-cyclodextrin, molar ratio 1:1.0.


Fig. 8. × , Clotrimazole g-cyclodextrin solubility diagram in microbiological growth medium at 37°C. Corresponding clotrimazoleand g-cyclodextrin concentrations during dissolution rate testing in the medium. , 7.65 mg/ml physical mixture clotrimazoleg-cyclodextrin, molar ratio 1:1.0; , 7.65 mg/ml inclusion complex clotrimazole g-cyclodextrin, molar ratio 1:1.0.

complex gave rise to in the Candida albicansgrowth medium was probably the reason why theinclusion complex had superior antimycotic effect,Fig. 5.

After the 24 h dissolution test of the cyclodex-trin inclusion complex, the solid phase in one ofthe tubes was isolated. After drying, HPLC analy-sis showed the content of clotrimazole was only22.1%. This is in agreement with the fact that onlya part of the g-cyclodextrin dissolved during thedissolution testing, Fig. 7. According to Fig. 5,the precipitate’s X-ray diffraction pattern differedfrom the pattern of the original clotrimazole g-cy-clodextrin inclusion complex.

3.4. Hemolysis and toxicity on TR146 cell culture

The toxicity of the clotrimazole inclusion com-plex, the physical mixture, clotrimazole and g-cy-clodextrin is compared in Figs. 9–11. Thehemolytic activity of the compounds correlatedwith the antimycotic activity, Fig. 9. The inclusioncomplex had the highest hemolytic activity fol-lowed by clotrimazole and the physical mixture.g-Cyclodextrin did not give hemolysis, Fig. 9. TheMTS/PMS toxicity assay on a monolayer ofTR146 cells (Jacobsen et al., 1996) showed thatthe inclusion complex was much more toxic thanthe other compounds tested, i.e. the physical mix-

ture, clotrimazole and g-cyclodextrin, Fig. 10.Due to the inclusion complex suspension’s ownabsorbance of light at 492 nm, the testing waslimited to 0.19 mg/ml. At 0.78 mg/ml and espe-cially at 3.1 mg/ml the light absorbance of thesuspended complex affected the resultsprofoundly.

Measurement of the transepithelial electrical re-sistance of multilayers of TR146 cells before andafter 4 h exposure to the compounds mentionedabove, i.e. 16 mg/ml inclusion complex, 16 mg/mlphysical mixture, 3.1 mg/ml clotrimazole and 12

Fig. 9. Hemolysis (%) of g-cyclodextrin 12 mg/ml; clotrimazole3.1 mg/ml; 16 mg/ml physical mixture clotrimazole g-cyclodex-trin, molar ratio 1:1.0; 16 mg/ml inclusion complex clotrima-zole g-cyclodextrin, molar ratio 1:1.0. Incubation time 15 min.


Fig. 10. MTS/PMS assay of TR146 cells testing of clotrima-zole and g-cyclodextrin compositions. The cellular sensitivity isexpressed as a function of the clotrimazole concentrationapplied. Average and S.E.M., n=8. The absorbance wasrecorded at 492 nm. , inclusion complex clotrimazole g-cy-clodextrin, molar ratio 1:1.0; + , clotrimazole; , physicalmixture clotrimazole g-cyclodextrin, molar ratio 1:1.0; ", neatg-cyclodextrin applied in concentrations corresponding to thecontent of cyclodextrin in the inclusion complex.

inclusion complex. After the inspection, theTR146 cell layers were trypsinated and the viabil-ity of the suspended cells was tested by trypanblue staining, Fig. 11. According to the results,the inclusion complex was not more toxic than theother compounds.

The results of the yeast and toxicity studiesindicate that the ability of the inclusion complexto cause clotrimazole supersaturation gave highactivity on fragile cells, i.e. Candida albicans cells,human erythrocytes and monolayers of not differ-entiated TR146 cells. The toxicity of the complexwas not detectable when a more robust cell sys-tem, i.e. multilayers of differentiated TR146 cells,was applied.

4. Conclusion

A genuine g-cyclodextrin inclusion complex ofclotrimazole, molar ratio 1:1.0, was isolated bycrystallization in phosphate buffer 0.05 M, pH7.1. The inclusion complex had superior in vitroantimycotic effect compared with clotrimazoleand a physical mixture of clotrimazole and g-cy-clodextrin. Probably, the superior effect wascaused by the complex’s ability to cause clotrima-zole supersaturation of the growth medium. Theclotrimazole supersaturation was probably alsothe reason why the complex had higher toxicitythan clotrimazole and the physical mixture onerythrocytes and on human TR146 cells.

Acknowledgements

The Danish Health Research Council, FertinLaboratories A/S and Ludvig Tegner og hustrusMindelegat are acknowledged for financial sup-port. Ms Jytte Davidsen is acknowledged for herskilful technical assistance.

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A genuine clotrimazole γ-cyclodextrin inclusion complex-isolation, antimycotic activity, toxicity...

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