Interaction between nitroheterocyclic compounds with β-cyclodextrins: Phase solubility and HPLC studies

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Interaction between nitroheterocyclic compounds with β-cyclodextrins: Phase solubility and HPLC studies

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   Journal of Pharmaceutical and Biomedical Analysis 47 (2008) 865–869 Contents lists available at ScienceDirect  Journal of Pharmaceutical and Biomedical Analysis  journal homepage: www.elsevier.com/locate/jpba Interaction between nitroheterocyclic compounds with  -cyclodextrins:Phase solubility and HPLC studies Nathalie F.S. de Melo a , Renato Grillo a , Andr´e Henrique Rosa a ,Leonardo Fernandes Fraceto a , b , ∗ a Departamento de Engenharia Ambiental, Universidade Estadual Paulista J´ulio de Mesquita Filho, Campus Sorocaba, Av. Trˆes de Marc¸o 511,CEP 18087-180, Sorocaba, S˜ao Paulo, Brazil b Departamento de Bioqu´ımica, Instituto de Biologia, Unicamp, Cidade Universit ´aria Zeferino Vaz s/n, Campinas, SP, Brazil a r t i c l e i n f o  Article history: Received 21 November 2007Received in revised form 15 April 2008Accepted 18 April 2008Available online 29 April 2008 Keywords: Nitroheterocyclic compoundsHydroxymethylnitrofurazoneCyclodextrinHPLCSolubility isotherm a b s t r a c t Chagas disease is a serious health problem for Latin America. Nitrofurazone (NF) and Hidroxymethyl-nitrofurazone (NFOH) are active against  Trypanosoma cruzi . The effect of    -cyclodextrin (  -CD) anddimethyl-  -cyclodextrin(DM-  -CD)complexationontheUVabsorptionandretentiontimeofnitrofura-zone(NF)anditshydroxymethylatedanalog(NFOH)werestudiedinsolution.Theretentionbehaviorwasanalyzed on a reversed phase C 18  column and the mobile phase used was acetonitrile-water (20/80 v/v),in which cyclodextrins (  -CD or DM-  -CD) were incorporated as a mobile phase additive. The decreasein the retention times of NF (or NFOH) with increasing concentration of HP-  -CD enables the determi-nation of the complex stability constants by HPLC. A phase-solubility study was performed, according tothe method reported by Higuchi and Connors, to evaluate the changes of NF/NFOH in the complexationstate,andthediagramsobtainedsuggestedthatitformscomplexeswithastoichiometryof1:1.Thisisanimportant study for the characterization of potential formulations to be used as therapeutic options forthe treatment of Chagas disease.© 2008 Elsevier B.V. All rights reserved. 1. Introduction Chagas disease affects about one quarter of the Latin Americapopulation. According to the World Health Organization, there areabout 120 million people living in risk of contracting parasitosisand 16–18 million people infected with the parasite [1]. The main problem with the treatment is the resistance of   Trypanosoma cruzi to drugs [2].New candidate drugs have been proposed for Chagas diseasechemotherapy and nitroheterocyclic compounds have been testedas antichagasic drugs to face this serious health problem in LatinAmerica. Nitrofurazone (NF, 5-nitro-2-furaldeyde semicarbazone,Fig.1)wassynthesizedbasedontheknowledgethatfuroicacid,aswell as its derivatives, demonstrates antimicrobial activity, beingactiveagainstGram-positiveandGram-negativebacteria[3].How- ever, its high toxicity has precluded its use in systemic infections[4]. In contrast, a report has shown that nitrofurazone is also abletodestroy T.cruzi throughtheinhibitionoftrypanotionereductase,an enzyme found in the parasite but not in the host [5]. ∗ Corresponding author. Tel.: +55 15 3238 3414; fax: +55 15 3228 2842. E-mail address:  leonardo@sorocaba.unesp.br (L.F. Fraceto). Hydroxymethylnitrofurazone (NFOH, Fig. 1) is one of the new candidate drugs for Chagas disease chemotherapy that showed tobe,  in vitro , very potent against  T. cruzi  [6].In the development of pharmaceutical products,   -cyclodextrins, a category of pharmaceutical excipients, havebeen widely used to improve solubility, chemical stability andbioavailability of a number of poorly soluble compounds.Cyclodextrins (CD) are cyclic oligosaccharides composed of glucopyranose units and can be represented as a truncatedcone structure with a hydrophobic cavity [7]. The cavity of  CDs is relatively hydrophobic compared to water, while theexternal faces are hydrophilic [8]. The most extraordinary characteristic of a cyclodextrin is its ability to form inclu-sion complexes with a variety of compounds, i.e., cagingforeign molecules (guest) through their interaction with theCD cavity in aqueous solution. It has been well estab-lished that the ability of    -cyclodextrins to enhance thestability and solubility of drugs is mediated through theformation of inclusion complexes [9]. Unmodified or unsub- stituted   -cyclodextrin, that is, with no substituent on theglucopyranose unit, presents poor water solubility and is par-enterally unsafe due to its nephrotoxicity. Therefore, severalsynthetically modified and relatively safe   -cyclodextrin, suchas hydroxypropyl-  -cyclodextrin, sulfobutylether-  -cyclodextrin, 0731-7085/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.jpba.2008.04.022  866  N.F.S. de Melo et al. / Journal of Pharmaceutical and Biomedical Analysis 47 (2008) 865–869 Fig. 1.  Chemical structure of NF, NFOH and schematic representation of   -cyclodextrins. dimethyl-  -cyclodextrin, have been produced and used in par-enteral formulations [10,11].In a previous work, we have investigated the interactionbetween NF [12] and NFOH [13] with 2-hydroxypropyl-  -CD.These studies showed that the formation of the inclusion complexchangedthesolubility,releaseprofileandthephotostabilityofbothNF and NFOH.The aim of the present study was to characterize the inclusioncomplexformedbetweenNForNFOHandcyclodextrins(  -CDandDM-  -CD) through the study of the HPLC retention behavior of NForNFOHinpresenceofcyclodextrinsandthroughphase-solubilityisotherms studies. This is an elementary study for the characteri-zationofapotentialformulationtobeusedasatherapeuticoptionfor Chagas disease. 2. Experimental  2.1. Reagents and chemicals Nitrofurazone and Hydroxymethylnitrofurazone were syn-thesized as previously described [6];   -CD and randomlymethylated-  -CD (degree of substitution ∼ 1.7–1.9) were obtainedasagiftfromRoquette.HPLC-gradeacetonitrile(ACN)wasobtainedfrom J.T. Baker and deionized water at 18m   from a Waters ultrapure water system. The solutions were filtered trough 0.22  mMillipore ® nylon membrane filter (Belford, USA).  2.2. Effect of cyclodextrin in NF/NFOH retention time by HPLC  The chromatographic experiments were performed using aShimadzu SCL-10VP controller pump, a Shimadzu SIL-10AD VPauto injector, a UV–vis SPD-10A VP detector (detection: 260nmfor NFOH) and Class-VP 6.12 as software. A reversed phase Phe-nomonex Gemini C 18 , 5  m, 10cm × 0.46cm was employed. Themobile phase used for these studies was acetonitrile–water (20/80v/v), in which cyclodextrin was dissolved (0, 5, 10, 15, 20, 30mM).The whole solution was filtered through a 0.2  m pore size nylonmembrane filter. The mobile phase was pumped at a flow rate of 1.0mL/min. The chromatographic experiments were carried outat 25 ◦ C. The NF/NFOH concentration in the injected solution was60  M and the injection volume was 0.2mL in all experiments.TheretentionbehaviorofNF/NFOHisgovernedbythedrugpar-titioncoefficientsbetweenthemobileandstationaryphases.Inthepresenceofcyclodextrins,thereisanadditionalcontributiontothedrug retention behavior due to the complexation process.The capacity factors for NF/NFOH were monitored in the pres-ence of increasing concentration of cyclodextrin. The stabilityconstant of the complex,  K  , was determined in triplicate, using Eq.(1) [14]:1 k ′  = 1 k ′ s + K  [CD]  x k ′ s (1)where  k ′ is the capacity factor at each cyclodextrin concentration[CD],and k ′ s  isthesolutecapacityfactorinabsenceofcyclodextrin.For a 1:1 stoichiometry complex, a plot of 1/ k ′ versus [CD] yields astraight line and K is obtained from the slope-to-intercept ratio.  2.3. Determination of the stability constants Excess amounts of NF/NFOH were added to 10mL glass tubescontainingdifferentconcentrationsofcyclodextrin.Thetubeswereshakenuntilequilibriumwasreached(32h)at25 ◦ C.Then,thesolu-tions were centrifuged at 25 ◦ C and the concentration of NF/NFOHwas spectrophotometrically determined at 260nm using a Femtospectrophotometer. The presence of cyclodextrin did not interferein the spectrophotometric assay of NF/NFOH. Deionized water was  N.F.S. de Melo et al. / Journal of Pharmaceutical and Biomedical Analysis 47 (2008) 865–869  867  Table 1 Apparent stability constants,  K  , for NF and NFOH with cyclodextrins inclusion complexes determined from HPLC study, 25 ◦ CInclusion complex Apparent stability constant (M − 1 ) Correlation coefficient,  r   Slope InterceptNF:b-CD 2.0  ±  0.3 0.997 2.52 1.241NF:DM-b-CD 16.8  ±  0.7 0.998 19.9 1.186NFOH:b-CD 1.6  ±  0.2 0.997 1.57 0.945NFOH:DM-b-CD 8.6  ±  0.1 0.998 8.03 0.924 usedasthebackgroundduringthespectrophotometricdetermina-tion of NF/NFOH concentration.When a linear relationship between the solubility of NF/NFOHand the concentration of cyclodextrin is obtained, the diagram isclassified as A L  , according to Higuchi and Connors [15] and theexperimental data fit equation: K   = slope S  o (1 − slope) (2)where  S o  is the molar solubility of NF/NFOH. The stability constantof the complex formed,  K  , can be obtained from the slope of thestraight line.A very reliable method for evaluating the solubilizing poten-tial of cyclodextrins is to determine the complexation efficiency(CE),whichreferstothecomplexed/freecyclodextrinratioandthatcan be obtained from the slope of their phase-solubility profile,according to Eq. (3) [16,17]. CE = S  o K  1:1  = [D/CD][CD]  = slope1 − slope (3)where[D/CD]istheconcentrationofdissolvedcomplex,[CD]istheconcentrationofdissolvedfreecyclodextrin.CEvaluescanbeusedto calculate the D:CD ratio, according to Eq. (4) [16,17]: D : CD = 1 :  1 + 1CE   (4) 3. Results and discussion  3.1. Chromatographic determination of the stability constant  When cyclodextrins are added to the mobile phase, soluteretention is driven by the drug partition between the mobile andstationary phases and the solute complexation with cyclodextrins.According to the solute retention time and the void time, capacityfactorswerecalculatedforeachsoluteinthepresenceofincreasingconcentrations of cyclodextrins. As expected, the retention timesdecreases as the concentration of cyclodextrin in the mobile phaseincreases due to the formation of the analyte–cyclodextrin com-plex, which enhances the guest solubility in the mobile phase andreducesitsresidencetimeinthecolumn[18,19].Anassayisshown as an example of NFOH in the presence of DM-  -CD (Fig. 2, varia- tionintheretentiontimesobtainedbyreplicationwasalwayslowerthan 1%).The above-mentioned formation constant for NF orNFOH:cyclodextrin complex was calculated according to Fig. 3(a) and (b) and Eq. (1). The linear relationship between 1/ k ′ and thecyclodextrin concentration (Fig. 3) with correlation coefficient higher than 0.99 indicates that the behavior of NF/NFOH is welldescribed by the model, assuming a 1:1 stoichiometry betweenthe guest and cyclodextrins [20,21].ThestabilityconstantsobtainedforNF/NFOHwithcyclodextrinsare shown in Table 1.As can be observed in Table 1, the stability constant of NF (or NFOH) with   -CD is lower than the observed for DM-  -CD;this result is reported in literature for other drug:cyclodextrinssystems [22,23]. The differences in stability constants observed between NF and NFOH with cyclodextrins can be associated withphysicochemical properties of these molecules, because, hydrox-ymethyl derivatives as NFOH ( S o =0.992mg/mL and log P   0.19) ismore hydrophilic than NF ( S o =0.657mg/mL and log P   0.23) [6].The same behavior was described in the literature for the inclu-sion complex between NF and NFOH with 2-hydroxypropyl-  -CD[12,13].  3.2. Determination of the stability constant by solubility isotherm All phase-solubility diagrams of NF or NFOH with   -CD andDM-  -CD,withintheconcentrationrangestudied,displayedatyp-ical A L   type diagram (i.e., linear increase of NF or NFOH solubilitywith increasing cyclodextrin concentration), consistent with a 1:1molecular complex formation for all cyclodextrins (Fig. 4). The result observed showed a linear behavior which is unequivocalfor all CDs studied ( r  =0.996 or better). The binding constant,  K  , of the complexes was calculated from the slopes of the linear phase-solubility plots according to the methodology described before.Results are summarized in Table 2.As shown in Table 1 and Fig. 3 (and Table 2 and Fig. 4), the binding constant determined for both compounds (NF and NFOH)with the CDs followed the rank order: DM-  -CD>  -CD, reflect-ing an enhancement on binding and solubility with the presenceof methylated groups, since these groups seems to be importantfor the binding of these compounds in the cyclodextrin cavity, asdescribed in literature [22–24]. The correlation between the sta- bility constant for NF/NFOH in DM-  -CD and   -CD and valuesdetermined for the interaction of these compounds with HP-  -CD [12,13] show that the association constant  K   increases in theorder HP-  -CD<  -CD<DM-  -CD. The complexation ability is sig-nificantly enhanced by methylation, which enlarges the CD cavity,makesitsenvironmentmorehydrophobicandfavorstheadaptabil-ity of the CD towards a guest, through an enhanced flexibility [25].Thesameresultshavebeenreportedinliteraturefortheinteractionof nitroimidazole compounds with these cyclodextrins [23].The differences of   K   measured by HPLC and phase-solubilityisotherm (for NF and NFOH) might be explained by the amountofacetonitrilepresentinthemobilephaseoftheHPLCmethodandthetimeneededbythecomplexformationtoreachequilibrium.Itis  Table 2 Apparent stability constants,  K  , for NF and NFOH with cyclodextrins inclusion complexes determined by phase-solubility techniques, 25 ◦ CInclusion complex Apparent stability constant (M − 1 ) Correlation coefficient,  r   Slope Intercept,  S o  ( × 10 − 3 ) CE Drug:CD ratioNF:  -CD 27.0  ±  2.5 0.996 0.0296 1.128 0.028 1:36NF:DM-  -CD 72.6  ±  1.5 0.998 0.0859 1.294 0.094 1:11NFOH:  -CD 24.7  ±  2.8 0.998 0.0342 1.434 0.035 1:29NFOH:DM-  -CD 58.6  ±  1.6 0.997 0.0783 1.450 0.084 1:12  868  N.F.S. de Melo et al. / Journal of Pharmaceutical and Biomedical Analysis 47 (2008) 865–869 Fig.2.  DecreaseinNFOHretentiontimeinthepresenceofincreasingconcentrationsofDM-  -CD(0,5,10,15,20,25mM)at25 ◦ C.Chromatographicconditions—column:Phenomenex C 18 , 5  m, 10cm × 0.46cm; mobile phase: acetonitrile/water (20/80v/v). Fig.3.  Plotof1/ k ′ vs.[cyclodextrin](  -CDandDM-  -CD)forNFOHandNFat25 ◦ C.Chromatographic conditions as described in Fig. 2. wellknownthattheadditionofacetonitrileormethanolmayhaveanegativeeffectoncomplexformationwithcyclodextrin.Forthesestudiedsystems,itseemsthattheadditionoforganicsolventsledtoadecreaseinthebindingconstants.Thereareseveralfactorswhichmay contribute to this decrease: firstly, the amount of organic sol-vent results in a less polar mobile phase, in which the non-polarsolutes become more soluble and as a consequence, the soluteaffinity for the hydrophobic cavity of cyclodextrins diminishesand part of the driving force for inclusion is removed. Secondly,a phenomenon of competition between the solute and the organicsolvent for binding cyclodextrins may occur, even though organicsolvent binds weakly to cyclodextrins [26]. However, instead the difference in size order of the  K   values determined by HPLC andsolubility isotherm for the inclusion complex between NF/NFOHwith cyclodextrins, the data present a good correlation coefficient( r  >0.9)whenthe K  valuesdeterminedbyHPLCwasplottedagainstthe  K   values determined from solubility isotherm.A more accurate method to determine the solubilization effi-ciency of cyclodextrins is to measure the complexation efficiency(CE), i.e., the concentration ratio between cyclodextrin in a com-plex and free cyclodextrin, and to study the influence of differentpharmaceutical excipients on the solubilization [17,27].FromEqs.(3)and(4)theCEvaluesanddrug:cyclodextrinratios for the NF/cyclodextrins and NFOH/cyclodextrins inclusion com-plex were determined, as shown in Table 2 [17,27]. The high CE  N.F.S. de Melo et al. / Journal of Pharmaceutical and Biomedical Analysis 47 (2008) 865–869  869 Fig.4.  Phase-solubilitydiagramofNFandNFOHwithcyclodextrins(  -CDandDM-  -CD) at 25 ◦ C. values obtained for DM-  -CD indicate that this cyclodextrin is abetter solubilizer than  -CD, as described by the authors [17,27].In addition, drug:cyclodextrin ratios of 1:12 and 1:11 were cal-culatedforNFandNFOH:DM-  -CDinclusioncomplex,respectivelyindicating that, on a 1:1 (NF/NFOH:DM-  -CD) complexation, justoneoutofthenumberofDM-  -CDmolecules(12forNFand11forNFOH) was forming an inclusion complex with drug [17,27]. 4. Conclusions This study showed the physicochemical characterization forthe inclusion complex between NF, NFOH and two different   -cyclodextrins (  -CD and DM-  -CD). The results indicated thatstable drug complexes were prepared at 1:1 molar ratio and thatthe complexation is driven by the physical chemical properties of the drug molecules. The characterization was investigated by theanalysisofstabilityconstantsdeterminedbytwodifferentmethods(solubilityisothermandbyHPLC).Thisstudyprovidesperspectivesfor future experiments using this inclusion complex of NF/NFOHwith cyclodextrins in order to verify its therapeutic efficacy.  Acknowledgments ThisresearchwassupportedbyFundac¸ ˜aodeAmparo `aPesquisado Estado de S˜ao Paulo-Fapesp (05/03045-9). R.G. (06/00787-7) isthe recipient of fellowship from Fapesp. The authors would like tothanks Dra. Carla M.S. Menezes and Dra. Elizabeth Igne Ferreira(FCF-Usp/S˜ao Paulo) for the synthesis of NFOH. 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