Effect of acidic ternary compounds on the formation of ...csps/JPPS7(3)/V.Barillaro/MICO.pdf · J...

J Pharm Pharmaceut Sci (www.ualberta.ca/~csps) 7(3):378-388, 2004

Corresponding Author: Valéry Barillaro, Laboratory of Pharmaceuti-cal Technology, Department of Pharmacy, University of Liège, CHU-Tour 4, Bat. B36, Avenue de l'Hôpital, 1, 4000 Liège 1, [email protected]

Effect of acidic ternary compounds on the formation of miconazole/cyclodextrin inclusion complexes by means of supercritical carbon dioxide.

Valéry Barillaro, Pascal Bertholet, Sandrine Henry de HassonvilleLaboratory of Pharmaceutical Technology, Department of Pharmacy, University of Liège, CHU-Tour 4, Bat. B36, Avenue de l'Hôpital, 1, 4000 Liège 1, BelgiumEric ZiemonsLaboratory of Analytical Chemistry, Department of Pharmacy, University of Liège, CHU-Tour 4, Bat. B36, Avenue de l'Hôpital, 1, 4000 Liège 1, BelgiumBrigitte Evrard, Luc Delattre, Géraldine PielLaboratory of Pharmaceutical Technology, Department of Pharmacy, University of Liège, CHU-Tour 4, Bat. B36, Avenue de l'Hôpital, 1, 4000 Liège 1, Belgium

Received 27 August 2004, Revised 3 November 2004, Accepted 15 November 2004, Published 30 November 2004

Abstract Purpose: The aim of the study is to evaluatethe effect of different acidic compounds on the inclu-sion of miconazole (MICO) in several cyclodextrins(CDs) using supercritical carbon dioxide (SCCO2) pro-cessing. Methods: Physical mixtures were processed bySCCO2 at 30 MPa, 125°C during 60 minutes in a staticmode to produce inclusion complexes. The inclusioncomplexes were characterized by differential solubil-ity, Fourier transform infrared spectroscopy (FT-IR)and dissolution test. Results: The best inclusion yieldswere achieved with the combination of MICO baseand HPγ CD with or without acids. Maleic and fumaricacids influenced the MICO inclusion differently infunction of their conformation. During the process, amiconazole salt was observed with maleic acid andcharacterized by thermal analysis and mass spectrome-try. The kinetics inclusion followed a saturation-typeshape curve. FT-IR confirmed the presence of genuineinclusion complexes. The complexes MICO base/HPγ CD/(L-tartaric acid) enhanced the dissolutionrates of MICO more than the corresponding physicalmixtures did. Lastly, the stability study revealed thatthe complexes were stable. Conclusions: The forma-tion of stable complexes between MICO and CDs ispossible using SCCO2. Moreover an acidic ternarycompound is able to modify the formation of the com-plex. The inclusion complexes, which show better dis-solution profiles than those with the correspondingphysical mixtures, could lead to an increase of the oralbioavailability of MICO.

INTRODUCTION

CDs have been used in pharmaceutical technology formany years as solubilizing agents1. Although CDs canincrease the aqueous solubility of drugs, many applica-tions need large amounts of CDs. But for several rea-sons, such as production costs and toxicity, CDamounts have to be reduced in pharmaceutical use. Toachieve this goal, several approaches can be considered.The first is the use of chemically modified CDs, whichpresent a higher solubility in water. The secondmethod consists in adding a water-soluble polymer,like PVP or methylcellulose, with the aim to increasethe solubility of both the complex and the drug itself2.The third method is the use of an acidic ternary com-pound. In the case of a basic drug, the acidic agent, forinstance, a hydroxyacid, promotes the solubilization ofthe guest molecule both by forming a salt and byincreasing the stability constant of the complex3. Thesame approach could be applied to an acidic drug byadding a basic ternary compound4.

SCCO2 has been successfully used for the preparationof inclusion complexes with CDs. This innovatingprocess allows the production of solid inclusion com-plexes without the use of water or organic solvents.Van Hees et al.5 were able to prepare inclusion com-plexes with piroxicam, an anti-inflammatory non steroi-dal drug, and βCD by processing a physical mixturewith SCCO2 (150°C, 45 MPa during 180 minutes). Thepresence of an inclusion compound was proved by DSCand FT-IR. More recently, Charoenchaitrakool et al. pro-duced inclusion complexes between ibuprofen andmethyl-βCD6. The ibuprofen content in the productcorresponded to a 1:1 complex, and DSC found nocrystalline ibuprofen.

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MICO (1-2-((2, 4-dichlorophenyl)-2-(2, 4-dichlorophe-nyl)-methoxy) ethyl)-1-imidazole) is an antifungal drugwhich presents poor water solubility. Its pharmacolog-ical indications are limited to the treatment of intesti-nal and dermatological mycosis. Previously, MICOwas also administered intravenously for the treatment ofsystemic fungal infections in the form of a micellarsolution, with the drawback that this solution contains asurfactant, polyoxyl-35 castor oil, able to induceanaphylactoid reactions. Several CDs (βCD, HPβCD,SBE-7-βCD, γ CD, HPγ CD…) have been used tocomplex MICO into their cavity7-9. The influence of aternary acidic compound in solution was studied andthe results showed a synergic effect between the CDsand the acid on the solubilization of MICO; this effectis independent of the pH value. This property allowedthe development of an intravenous non-surfactantsolution10.

In this study, the effect of both the CD type and theacidic ternary agent on the formation of solid inclusioncomplexes with MICO by means of SCCO2 will beinvestigated. In a second step, the influence of the con-formation of the ternary compound will be deter-mined. In a third step, the effect of process time on theinclusion will be studied. Then, the formation of inclu-sion complexes will be confirmed by FT-IR spectros-copy, differential solubility and dissolution studies.

EXPERIMENTAL SECTION

Materials

MICO (m.p. 84.3 °C, Eur. Ph. 4th Edition) wasobtained from Janssen Pharmaceutica (Beerse, Bel-gium). MICO nitrate (m.p. 184.1°C, Eur. Ph. 4th Edi-tion) was purchased from Bufa (Uitgeest, Holland), βCD(Eur. Ph. 4th Edition, 7.58% H2O) and HPβCD (3.22% H2O, D.S. 0.63) were kindly supplied by Roquette(Lestrem, France). γ CD (4.25 % H2O) and HPγ CD(1.62 % H2O, D.S. 0.58) were obtained from WackerChemie GmbH (Munich, Germany). Citric acid (m.p.153°C, pKa1 3.13, pKa2 4.76, pKa3 6.40, Eur Ph. 4th

Edition) and L-tartaric acid (m.p. 206°C, pKa1 2.93,pKa2 4.23, Eur Ph. 4th Edition) were purchased fromMerck (Darmstadt, Germany), malic acid (m.p. 131°C,pKa1 3.46, pKa2 5.10) from Sigma-Aldrich (Steinhem,Germany), fumaric acid (m.p. 287 °C, pKa1 3.10, pKa27.80) from Fluka (Buchs, Switzerland) and maleic acid(m.p. 139°C, pKa1 1.90, pKa2 6.70) from Acros (New

Jersey, USA). The chemical structure of all these com-pounds is depicted in figure 1.

Figure 1: Chemical structures of MICO and the usedacids.

R14821 (2, 4-dichlorophenyl)-1H-imidazol-1-ethanol)were purchased from Sigma-Aldrich (Steihnhem, Ger-many) and R66716 (2, 4-dichlorobenzenemethanol)from Acros (New Jersey, USA). CO2 was of N48 qual-ity from Air Liquide (99.998%) (Liège, Belgium). Allother products were of analytical grade.

Methods

Preparation of physical mixtures

The physical mixtures were prepared by grinding, in amortar, calculated and exactly weighed amounts ofMICO, CDs and acid in equimolar ratio.

Preparation of inclusion complexes

The inclusion experiments with SCCO2 were per-formed with a SUPREX SF Extractor Autoprep 44(Pittsburgh, PA, USA), described in previous works5.A 1-ml vessel was filled with 400 or 600 mg of physicalmixtures of MICO-CD 1:1 (mol:mol) or MICO-CD-acid 1:1:1 (mol:mol:mol). The content was pressurized(± 1 minute) and left in a static mode during 60 min-

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utes at 30 MPa and 125°C. At the end of the experi-ment, the vessel was depressurized within 15 seconds.The vessel content, in the form of a compact solid, wasemptied, ground and homogenized in a mortar beforefurther analysis. The kinetics of the formation of theinclusion complexes were also studied by processingphysical mixtures with SCCO2 as described above dur-ing various contact times (15, 30, 60, 120 and 240 min-utes). The effect of an acidic ternary compound wasalso determined.

Assay of miconazole

MICO was assayed by an HPLC method. The HPLCsystem consisted of an L-6000 Merck-Hitachi pressurepump, an L-7200 Merck-Hitachi autosampler, an L-7350 Merck-Hitachi column oven, an L-7400 Merck-Hitachi UV detector and a D-7000 interface. The sys-tem was controlled by a computer running the “HPLCSystem Manager v 4.0” acquisition software developedby Merck-Hitachi. Twenty-µl samples were injectedinto a Lichrocart column (125 x 4 mm i.d.) preparedwith an octylsilane (C8) phase Lichrospher 60 RP-Select B 5 µm (Merck) and maintained at 30°C. Themobile phase consisted of a 70:30 (v/v) mixture ofmethanol (HPLC grade) and a 50 mM pH 3.5 ammo-nium acetate buffer. The flow rate was adjusted to 1.0ml/min. All samples were analyzed twice. The detec-tor was set at 230 nm. This method was validated11 andshowed good linearity, reproducibility and accuracybetween 5 and 80 µg/ml. Limits of detection (LOD)and quantification (LOQ) were determined and foundto be 0.4 µg/ml for the LOD and 1.33 µg/ml for theLOQ.

Determination of inclusion yields

The inclusion yields were determined by a differentialsolubility method according to Van Hees et al.12. Thismethod includes two steps. The first consists in the sol-ubilization of the physical mixture or the complex in asolvent that dissolves the MICO, the CD and the ter-nary agent. This step allows determining total MICOcontent. The second step consists in the solubilizationof the free MICO with an organic solvent in which theCD is insoluble and in which the included drugremains entrapped. So the total MICO content wasdetermined by dissolving an exactly known quantityof MICO complex or physical mixture in the HPLCmobile phase. The MICO contained in the resulting

solution was assayed using the HPLC methoddescribed above. In the same way, the free MICO con-tent was measured by dispersing an exactly knownquantity of MICO complex or physical mixture in ace-tonitrile. The suspension was sonicated (TranssonicT460) for five minutes, filtered through a Millex-GV0.22 µm filter (Micropore) and analyzed by HPLCafter appropriate dilution in order to determine thefree MICO concentration. Determination of the totalMICO content in the complexes produced by super-critical processing allows assessing that there is no lossof MICO during this process. In order to validate thedifferential solubility method, physical mixturesbetween MICO base and βCD, and between MICObase and γ CD, were prepared by gently grinding thepowders in a mortar with a pestle. For each combina-tion, the MICO base concentrations were 1.79 % (w/w) and 28.57 % (w/w) to reach final concentrations of5 and 80 µg/ml of MICO base respectively. Two ref-erence inclusion complexes were prepared. The firstwas a MICO base/βCD inclusion complex obtained bya precipitation method described by Pedersen et al.8. 16mg/ml βCD were dissolved and 0.4 mg/ml MICO basewere dispersed in a 50 mM pH 10 phosphate buffer. Themixture was stirred during 15 days at 4°C. After thistime, the suspension was filtered and the insoluble frac-tion was kept in an oven at 60°C until dryness. The sec-ond complex was a MICO base/γ CD inclusioncomplex prepared according to Jacobsen et al.9. 100mg/ml γ CD were dissolved and 1 mg/ml MICO basewas dispersed in a 50 mM pH 7 phosphate buffer. Thesuspension was stirred at room temperature during 15days. After this time, the suspension was filtered andthe insoluble fraction was kept in an oven at 60°Cuntil dryness. The complete MICO inclusion in bothcomplexes was checked by DSC.

Infrared spectroscopy

The spectra of MICO, CDs, acids, physical mixturesand complexes were obtained from a KBr disk with aPerkin-Elmer GX FT-IR spectrophotometer (Beacons-field, United Kingdom). The frequency range liesbetween 4000 and 450 cm-1.

Differential scanning calorimetry (DSC)

DSC experiments were carried out with a Mettler-Toledo DSC25-TC15 TA controller supervised by acomputer running the “Star System v.6.10 SW” soft-

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ware. The experimental conditions were as follows:temperature range from 30 to 250°C with a heatingrate of 10°C/min, pierced aluminum crucibles withabout 6 mg product, nitrogen atmosphere with a 20ml/min flow rate.

Liquid chromatography-Mass spectrometry (LC-MS) condi-tions

The HPLC is an HP 1000 series consisting of a binarypump, a vacuum degasser, a thermostated columncompartment and an autosampler, all from AgilentTechnologies (Waldbronn, Germany). The HPLC sep-arations were performed at 30°C on a Waters RP-C8Symmetry column (5 µm, 125 x 4 mm i.d.) (Milford,Massachusetts, U.S.A.). The mobile phase was a mix-ture of methanol and of a 50 mM pH 3.5 ammoniumacetate buffer (70:30 v/v). The flow rate was 1 ml/min.and the injected sample volume was 20 µl. Mass spec-trometry detection was carried out using a MicromassUltima Quadrupole instrument (Manchester, UnitedKingdom) configured with a Z-spray electrospray ion-ization source and controlled by a computer runningthe “Masslynx v. 3.5” acquisition software. Source con-ditions were as follows: positive ion electrospray; capil-lary voltage: 3 kV; cone voltage: 14 V; sourcetemperature: 145°C; desolvatation temperature:450°C; cone gas flow (nitrogen): 94 l/h; and desolvata-tion gas flow (nitrogen): 552 l/h. Sample solutionswere prepared by dissolving the compound in themobile phase to reach the concentration of 10 µg/ml.

Dissolution studies

The dissolution profiles of MICO, physical mixturesand complexes were performed according to the rotat-ing paddle method described in the 4th Edition of theEuropean Pharmacopoeia. A dissolution test appara-tus, Sotax AT7 (Basel, Switzerland), connected via aWaston-Marlow 505Du peristaltic pump (Falmouth,United Kingdom) to a Hitachi 3000 flow-through UVspectrophotometer (Tokyo, Japan) was used in thismethod. A quantity equivalent to 13 mg of MICO basewas exactly weighed, introduced into a hard gelatincapsule, and then immersed by means of a sinker into900 ml of dissolution medium containing a 50 mM pH6.8 phosphate buffer. This medium was stirred at 100rpm and set at 37 ± 0.1°C. The medium was pumpedat a rate of 15 ml/min every 2 minutes, passed througha 0.47-µm filter and the absorbance of this solution was

measured at 230 nm. Each experiment was carried outin duplicate. The standard solution at the concentra-tion of 25 µg/ml was prepared by dissolving MICObase in a 50/50 (v/v) methanol/50 mM pH 6.8 phos-phate buffer solution. The pH of the medium waschecked at the end of the experiment.

Stability studies

The stability of the inclusion yield of the complexesproduced by SCCO2 was determined. Three combina-tions were studied: the complexes between MICO base/HPγCD, MICO base/HPγ CD/citric acid and MICO base/HPγCD/L-tartaric acid and the corresponding physicalmixtures.

The samples were placed in airtight glass containers andstored in a Binder KBF environmental test chamber(Tuttlingen, Germany) at 25°C ± 1°C. All sampleswere stored in triplicate. The total and free MICO con-tents were determined in both complexes and physicalmixtures after 0, 7, 15, 30, 60, 120, 180, 270 and 360days of storage.

RESULTS AND DISCUSSION

Validation of the differential solubility method

Determination of extraction efficiency

Results of extraction recoveries are presented in table 1.

Table 1: Determination of the extraction efficiency ofMICO in physical mixtures with βCD and γ CD.

For all physical mixtures, as extraction efficiencies arehigher than 94%, the extraction can be considered ascomplete. The calculated inclusion yields are lowerthan 2.62%. Since all the MICO is free in physical mix-tures, the obtained inclusion values confirm that thisdifferential solubility method is able to solubilize allthe free MICO.

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Analysis of the precipitated miconazole/CD inclusioncomplexes

The analysis of reference inclusion complexes betweenMICO and CD using the differential solubility methodand the DSC are given in table 2.

Table 2: Differential solubility analysis and DSC analysisof precipitated MICO inclusion complexes.

In these samples, MICO is completely included in theCD cavity and the free MICO content is equal to zero.The results for the MICO/βCD complex analysis showan inclusion yield of 99.19%, thus confirming the DSCresults. For the MICO/γ CD complex, the measuredinclusion yield is 88.67%. The observed difference inthe free MICO content between the DSC and the differ-ential solubility method is the result of a pseudopoly-morphic transformation of MICO during thepreparation of the inclusion complex13. Indeed, MICOcan precipitate onto γ CD particles in an amorphousform, which is considered as free MICO by the differen-tial solubility method, but not detected by thermalanalysis (DSC). In conclusion, the differential solubil-ity method is able to give an accurate determination ofthe inclusion yield in MICO/CD complexes.

Effect of both cyclodextrins and acids on the complex formation

Effect of both cyclodextrins and hydroxyacids on the inclu-sion process

As shown in table 3 for the binary mixtures, no signifi-cant inclusion occurs with βCD and HPβCD for either ofthe two MICO types.

The maximum yield, which lies at about 37%, isobtained with the MICO base/HPγ CD combination.Table 3 also shows the influence of five different acidson MICO inclusion (respectively for citric, DL-malic, L-tartaric, maleic and fumaric acids). Compared to binarysystems, the yields are remarkably modified by the pres-ence of these acids. Moreover, the use of a hydroxypro-pylated CD gives better results than those obtainedwith the native CD. As in the binary complex, theinclusion with βCD is insignificant.

Table 3: MICO inclusion yields in function of MICO, CDand acid types (n = 3).

For the ternary mixture containing MICO base, HPγ CDand citric acid, the inclusion yield is close to 80%, whilefor the same combination with DL-malic or L-tartaricacid, the inclusion is about 90%. These acidic com-pounds multiply the inclusion yield by two. HPβCDgives similar results. In combination with MICO baseand citric or DL-malic acid, the inclusion is close to50%, whereas L-tartaric acid gives an inclusion yield ofaround 90%. At this point in the study, several factorsthat are important for inclusion can be pointed out:the use of MICO base, the presence of both a hydroxy-acid (especially L-tartaric acid) and a hydroxypropy-lated CD, and the CD cavity size. Firstly, in allinclusion experiments, MICO base gives better resultsthan the nitrate does. This could be a result of the dif-fusion coefficient of MICO in the SCCO2. Indeed, thediffusion coefficient of a guest molecule in the SCmedium influences the interactions between the guestmolecule and the CD. Data on the diffusion coeffi-cients of MICO base and nitrate in SCCO2 are notavailable. As SCCO2 presents a poor dielectric con-stant, ranging from ~ 1 to 2 in function of pressureand temperature14, MICO nitrate should not be disso-ciated in this medium. Consequently, its diffusion coef-ficient, which also depends on molecular shape15,should be lower than that of the MICO base, whichpresents a more compact shape than MICO nitrate’s.Moreover, the interactions between the guest and thehost are promoted when the guest is not protonatedbecause the polarity of the CD cavity looks like thepolarity of a hydro-alcoholic solution16. MICO type

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also has an effect on the interaction with the acid.Indeed, as mentioned in the literature, the acidic ter-nary agent interacts with a basic function of the guestand with the hydroxyl group of the CD17. In this case,hydroxyacid does not fit into the CD cavity, butremains outside, while MICO forms an inclusion com-plex through the interaction between its O-CH2-dichlorobenzene group and the CD cavity18. Electro-static interactions between the acid and the imidazolering of the drug take place on one end. Hydrogenbonds with the hydroxyl group of the CD are formedon the other end. So, by bridging the guest and thehost, the acid has two interaction points. In the case ofMICO nitrate, the imidazole ring is already protonatedand cannot interact with the acid by formation ofhydrogen bonds. Moreover, the acidic constant ofnitric acid (pKa –1.40) is smaller than the pKa of thehydroxyacids, which are not able to shift the nitricacid, thereby limiting the interactions MICO-acid.This is the reason why the yields are not modified verymuch by the presence of an acidic ternary compound.Secondly, the modified groups on the CD ring have apositive influence on the formation of hydrogen bondsbetween the acid and the CD. As reported by Zia etal.19, the complexation properties of the CD are modi-fied by the hydroxypropyl substitution. The cavity ofthe CD seems to be enlarged and new hydrogen bondscan occur between the guest and the host in order tostabilize the formed complex. Lastly, by referring to thesolid-state structure of the γCD, the crystallization waterincluded in the cavity is characterized by a high degreeof disorder, greater than in the βCD. Some of thesewater molecules seem to be in an activated state whichcould promote the formation of inclusioncomplexes20. This energetically favorable state couldexplain why inclusion is not possible with the βCD inthe supercritical conditions tested.

Effect of the conformation of the ternary compound

In order to estimate the effect of the acid conforma-tion, the influence of fumaric and maleic acid was eval-uated. The data are also reported in table 3. These acidsincrease the inclusion in the same way as the hydroxy-acids, but the effect depends on the conformation ofthe double bond. When the double bond conformationis cis, as for maleic acid, the interactions are higherthan when the conformation is trans, like for fumaricacid, except for βCD. So, for the MICO base/HPγ CD/

maleic acid complex, the inclusion yield is about 58%,while it is 32% with fumaric acid. This kind of molecu-lar interaction has been described by Muñoz de la Peñaet al.21. By forming ternary complexes between βCD,pyrene and several alcohols, they demonstrated that thesize and conformation of the alcohol lead to larger equi-librium constant values for the complexes when there isa good matching among the three inclusion complexcomponents.

Differential solubility studies reveal the decrease ofMICO in the maleic acid complexes, to the benefit ofanother compound. It was shown that this compoundcorresponds to the formation of MICO maleate.

Figure 2a depicts the analysis of total MICO contentby differential solubility for a MICO base/HPγ CD/maleic acid complex.

Figure 2: HPLC Chomatogram obtained after the analysisof total MICO content for the MICO base/HPγ CD/maleicacid SCCO2 complex (a) and the analysis of R14821,R66716 and MICO base at equal concentration (40 µg/ml) (b).

It shows the presence of MICO base at 5.97 minutesretention time and also the presence of another com-pound at 3.33 minutes retention time that is not theresult of the typical degradation products of MICObase in acidic conditions, i.e. R14821 and R66716.Indeed, the analysis of methanolic solution of R14821,R66716 and MICO base at 40 µg/ml concentrationreveals following retention times: 1.43, 2.43 and 5.97minutes respectively (figure 2b).

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A physical mixture of MICO base and maleic acid wasprepared in equimolar proportions and left in an ovenat 125°C for 60 minutes, these conditions simulatingtemperature conditions in the SCCO2 experiment. Inthese circumstances, the MICO base melts (m.p. =84.3°C) and reacts with maleic acid (m.p. = 139°C) toform MICO maleate. The resulting product shows thesame retention time as the product formed after treat-ment with SCCO2 (data not shown). DSC analyseswere performed.

Figure 3 displays the DSC curves of the MICO base,the physical mixture between MICO, and maleic acidand MICO maleate.

Figure 3: DSC profiles of MICO base (a), physical mixtureof MICO base/maleic acid (b) and MICO maleate (c).

The DSC profile of the physical mixture can bedivided into different steps: the melting of the MICObase, the melting of maleic acid, an exothermic phe-nomenon (at 130°C) which is attributed to the salt for-mation and its crystallization, and finally, the saltmelting point at 210°C. Analog thermal behavior hasbeen previously described by Mura et al. with an acidicdrug in the presence of a basic aminoacid22. Lastly, LC-MS analysis was performed to confirm the presence ofsuch a salt. Figure 4 presents the results obtained dur-ing the LC-MS analysis of MICO maleate produced asdescribed above.

On the first chromatogram (figure 4a), the detection isset at m/z equal to 417 and MICO is observed at the5.15 minutes retention time. On the second chromato-gram (figure 4b), where the detection is set at m/zequal to 533, a product is observed at the retention

time of 3.00 minutes. The mass of this product corre-sponds to the addition of the molecular masses ofMICO base (MM = 416) and of maleic acid (MM =116), which confirms the presence of a MICO base salt:MICO maleate. The chromatogram obtained underbase peak ion (BPI) scan mode (figure 4c) confirms thegood separation of the compounds.

Figure 4: LC-MS chromatograms of a MICO maleatesolution: detection set at m/z 417 (a), detection set at m/z 533 (b) and base peak ion (BPI) scan mode (c).

This experiment allows to clearly identifying the com-pound observed during the analysis of the complexescontaining maleic acid by differential solubility (figure3b). So the compound detected at the 3.33 minutesretention time was MICO maleate. In conclusion, dur-ing the formation of inclusion complexes betweenCDs, MICO and maleic acid, two phenomena occur.The first is the formation of a ternary complexbetween the three compounds, and the second is theformation of a salt between MICO base and maleicacid.

Effect of the process time

The formation kinetics of an inclusion complexbetween MICO and HPγ CD were determined in thepresence and in the absence of citric acid. Results are

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shown in figure 5. Kinetics profiles of MICO inclusionshow a hyperbola shape. In both cases, a plateau isreached after a contact time of 60 minutes. At thistime, the inclusion yields are about 37% for the binarymixture and about 81% for the ternary one. So it canbe said that the acid does not modify the kinetics ofinclusion since the plateau appears at the same contacttime, but that it does modify the position of the com-plexation equilibrium, promoting the formation of theinclusion compound.

Figure 5: Kinetics of the complex formation by SCCO2 forMICO base into HPγ CD (■) and into HPγ CD in thepresence of citric acid (▲) at 30 MPa and 125° C.

Characterization of the complexes

FT-IR spectroscopy

Previous FT-IR analysis has shown that SCCO2 has noinfluence on the pure compound spectra (data notshown). The typical stretching bands of MICO basewere found at 1562 and 1589 cm-1 (dichlorosubstitutedbenzene) and at 1468, 1506 and 1546 cm-1 (imidazolering). These bands appear unchanged in the physicalmixtures (figure 6).

However, shifts and attenuations of these characteristicbands show that there are some interactions betweenthe MICO and the CD by formation of an inclusion com-plex (table 4). The comparison of the FT-IR spectra ofthe MICO base/HPγCD/citric acid physical mixture (fig-ure 6e), the complex (figure 6f) and the MICO/citricacid compound (figure 6g) produced with the samemethod as MICO maleate, shows some modificationsof the typical MICO bands.

Figure 6: FT-IR spectra of MICO base/CD systems: MICObase (a), physical mixture with γ CD (1:1) (b), MICO/γ CD complex produced by SCCO2 processing (c) andMICO/γ CD complex obtained by precipitation (d), MICObase/HPγ CD/citric acid (1:1:1) physical mixture (e),MICO/HPγ CD/citric acid complex produced by SCCO2processing (f) and MICO/citric acid compound (g).

Table 4: Characteristic FT-IR bands positions of MICO inseveral different systems.

Moreover, a broad absorption band can be observed at1719 cm-1. This signal, not visible in the physical mix-ture, is attributed to a carboxylic C=O stretching thatgenerally occurs at 1725 cm-1, while the carboxylateC=O stretching typically occurs at around 1600 cm-1.

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This signal is amplified by an important interactionbetween the acid and the imidazole ring of the MICO.This explains why this function is visible in the com-plex, but not in the corresponding physical mixture.All these observations suggest that MICO interactswith the CD in order to form a genuine inclusion com-plex, and with the acid, which reinforces the associa-tion between the guest and the CD.

Dissolution studies

The dissolution profiles of MICO and of differentMICO/CD formulations are depicted in figure 7.

Figure 7: Dissolution curves of MICO base (▼), physicalmixture of MICO base/HPγ CD (■), MICO base/HPγ CDcomplex (▲), physical mixture of MICO base/HPγ CD/L-tartaric acid (◆), MICO base/HPγ CD/L-tartaric acidcomplex (●).

Before comparing the dissolution curves, it is interest-ing to note that the pH value of the dissolutionmedium remained unchanged after the different runs.It can be observed that the amount of dissolved MICOfrom the powders containing cyclodextrins is higherthan when the drug is alone. The physical mixturesshow evidence of faster dissolution rates than theMICO alone. The complexes give the largest improve-ment in drug dissolution, particularly the complexcontaining L-tartaric acid. The modification of theMICO dissolution in the physical mixture is the resultof interaction between the drug and the CD by the for-mation of a soluble inclusion complex in the dissolu-tion medium. Moreover, an acidic micro-pH, favorableto the drug dissolution, is formed thanks to the solubi-

lization of L-tartaric acid. As expected, the complexesformed by SCCO2 processing present better dissolu-tion profiles than the physical mixtures or the MICOalone. The best performance is obtained with the ternarycomplex between MICO base/HPγCD/L-tartaric acid.This complex already had the highest inclusion yield dueto the presence of L-tartaric acid, which seems to pro-mote dissolution. Finally, at the end of the dissolutiontest, a decrease in the MICO concentration is observedfor the ternary complex. This decrease in solubility is aresult of the dilution of the complex, which promotesthe dissociation of MICO from its inclusion com-pound, according to the binding constant value. SoMICO precipitates to reach the solubility of MICO atthe equilibrium in each final medium.

Stability study

The results obtained during the long-term conserva-tion of the complexes are presented in figure 8.

Figure 8: Long-term stability of the MICO base/HPγ CD(■), MICO base/HPγ CD/citric acid (▼) and MICO/HPγ CD/L-tartaric acid (▲) complexes.

In the physical mixtures, no significant inclusion yieldoccurs (data not shown). Statistical analysis was per-formed in order to estimate the effect of time onMICO inclusion. The mean values of the inclusionyields for each complex were compared by a one-wayvariance analysis at the p = 0.05 significance level. Theresults show that there is no significant modification inthe inclusion yield of MICO base for the MICO base/HPγ CD complex (p = 0.15), for the MICO base/HPγ CD/citric acid complex (p = 0.07) and for theMICO base/HPγ CD/L-tartaric acid complex (p = 0.13).So it can be concluded that the complexes are stable dur-ing the conservation period tested, i.e. one year.

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CONCLUSIONS

The study strongly suggests that it is possible to pro-duce genuine inclusion complexes between MICO andCD by SCCO2 processing. Some factors have a posi-tive influence on the inclusion yield, such as the typeof CD (CD derivatives and larger cavity size) and thetype of MICO (MICO base which possesses a higherdiffusion coefficient in SCCO2, in comparison with thenitrate). The addition of an acidic ternary compound dra-matically improves inclusion yield. The best results areobtained with the combination of MICO base, HPγCDand DL-malic or L-tartaric acids, and with the combina-tion of MICO base, HPβCD and L-tartaric acid, forwhich the inclusion reaction is nearly complete. In theseternary complexes, both MICO and CD types influencethe drug inclusion, like they do in binary complexes. Thestudy of the kinetics for the MICO base inclusion intoHPγ CD reveals that the time course of inclusion dis-plays a saturation-type shape curve and that the acidiccompound does not modify the kinetics, but ratherpromotes the inclusion of the drug. Moreover, the con-formation of the ternary compound is an importantfactor because it influences the interaction betweenguest and host. Stability studies reveal that the com-plexes indeed present an attractive long-term stabilityup to one year. During the analysis of the complexescontaining MICO base and maleic acid, the presence ofa salt, MICO maleate, has been found. It has been char-acterized by DSC and LC-MS. FT-IR analysis con-firmed the existence of genuine MICO/CD inclusioncompounds, and the dissolution studies showed thatthe complexes are more soluble than either the MICOalone or the corresponding physical mixtures. Thisproperty could be interesting for use of the complexesin a pharmaceutical dosage form, with a view toimproving the drug’s bioavailability after oral adminis-tration.

ACKNOWLEDGEMENTS

G. Piel is a postdoctoral researcher supported by theF.N.R.S., Brussels, Belgium.

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