1 H NMR studies of binary and ternary dapsone supramolecular complexes with different drug carriers:...

Research article

Received: 20 November 2013 Revised: 7 May 2014 Accepted: 10 May 2014 Published online in Wiley Online Library: 14 July 2014

(wileyonlinelibrary.com) DOI 10.1002/mrc.4087

1H NMR studies of binary and ternary dapsonesupramolecular complexes with different drugcarriers: EPC liposome, SBE-β-CD and β-CDLucas Martins,a Monica Arrais,b Alexandre de Souzab and Anita Marsaiolia*

Binary and ternary systems composed of dapsone, sulfobutylether-β-cyclodextrin (SBE-β-CD), β-CD and egg phosphatidylcholine(EPC) were evaluated using 1D ROESY, saturation transfer difference NMR and diffusion experiments (DOSY) revealing the binarycomplexes Dap/β-CD (Ka 1396 lmol�1), Dap/SBE-β-CD (Ka 246 lmol�1), Dap/EPC (Ka 84 lmol�1) and the ternary complex Dap/β-CD/EPC (Ka 18 lmol�1) in which dapsone is more soluble. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords: NMR; dapsone; EPC liposome; SBE-β-CD; β-CD

* Correspondence to: Anita Marsaioli, Chemistry Institute, State University ofCampinas, Campinas, Brazil. E-mail: [email protected]

a University of Campinas, Chemistry Institute, Campinas, Brazil

b Federal University of Piauí, Post-Graduate Program in Pharmaceutics Sciences,Teresina, Brazil

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Introduction

Diaminodiphenylsulfone or Dapsone (Dap) has bacteriostaticactivity against Mycobacterium leprae and Pneumocystis carinii andis used in the treatment of infectious diseases, such as leprosyand pneumonia, in patients with AIDS. The suggested mechanismof action is varied and may involve inhibition of folic acid synthesisor act as a competitive antagonist of p-aminobenzoic acid.[1] Dap isadministered alone or combined with other drugs; however, Dap’slow solubility in water and in alcohol requires good pharmaceuticalformulations for clinical applications.[2] Drug water solubility can bealtered through supramolecular interactions using drug carriers,such as cucurbiturils[3–7], cyclodextrins,[8–14] calixarenes,[14–20] lipo-somes,[21,22] micelles,[23] solid lipid nanoparticles,[24,25] microparti-cles[26] and nanoparticles.[27] Dap and β-cyclodextrin (β-CD)interactions have been studied by applying different methodolo-gies, and their apparent association constants in the presenceand absence of various linear alcohols were determined byspectrofluorimetry.[28] Dap-β-CD complex stoichiometry has beenreported,[29] and several structural models were proposed basedon emission spectroscopy[30] and nuclear magnetic resonance[31,32]

(NMR) experiments. Additionally, ternary systems composed ofDap, polyvinylpyrrolidone K30 or hydroxypropyl methylcellulose,and β-CD or 1-HP-β-CD were investigated to improve Dap solubilityand bioavailability.[32] Liposomes have been extensively applied asdrug carriers, particularly egg phosphatidylcholine liposome (EPCL),which, together with β-CD and the local anaesthetic proparacaine,make stable ternary complexes.[33,34]

Notwithstanding the previously mentioned investigations, theinteraction between the liposomes of EPC and the Dap systemin binary and ternary complexes was not studied previously.Evaluation of Dap (1) binary complexes with different drug car-riers [sulfobutylether-β-CD (SBE- β-CD, 3), tert-butyl-calix[4]arene(calix[4], 4) and EPCL 400nm (EPCL 5)] applying different NMR tech-niques, such as rotating frame nuclear overhauser effect spectros-copy (1D ROESY),[35] saturation transfer difference (STD)[36–38] andmeasurements of the longitudinal relaxation rates and the molecu-lar diffusion constants, revealed the structures of the molecules,

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which are depicted in Scheme 1.[39–41] The same NMR spectro-scopic tools were subsequently applied to investigate the ternarycomplex composed of Dap, EPCL and β-CD (2).

Results and Discussion1H NMR chemical shift is a local parameter, and the observedeffects may result from a variation of chemical environmentrather than interaction forces. The chemical shifts can be thebasis for the determination of the strength of interaction onlythrough titration experiments and subsequent fitting of thelinear or nonlinear dependence of chemical shifts from the molarratios or total concentration. However, our aim to evaluate thechange in chemical shift is only qualitative because the strengthof the interaction was studied from variation in the diffusioncoefficient of substances. The Dap/β-CD complex has beenpreviously characterised;[28–32] however, its characterisation onthe same experimental conditions used for all drug carriersaddressed in this study was important for better data com-parison. Moreover, these data have to be consistent for a betterunderstanding of the ternary complex formation containingDap, EPCL and β-CD. Concerning the β-CD and SBE-β-CD(complexes Dap/β-CD and Dap/SBE-β-CD, respectively), chemicalshift variations were observed for Ha and Hb (Δδ=0.02 ppm inboth cases). Calixarene (4) is rather insoluble in D2O, D2O with20% DMSO-d6 and EPCL in D2O; therefore, attempts to

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Scheme 1. Chemical structures of dapsone (Dap, 1), sulfobutylether-β-cyclodextrin (SBE-β-CD, 3), tert-butyl-calix[4]arene (calix[4], 4), egg phosphatidyl-choline (EPC, 5 with a representative structures of EPC liposome) and β-cyclodextrin (β-CD, 2).

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encapsulate Dap in calix[4]arene used CDCl3 as the solvent, and nochemical shift variation was observed, indicating the absence of theDap/calix[4] complex formation in this solvent. With respect to theDap/EPC complex in D2O, chemical shift variations ofΔδ=0.07 ppmand Δδ= 0.06 ppm were observed for Ha and Hb, respectively.The greater variations in the chemical shifts of Dap comparedwith the variations occurring in the samples containing β-CDand SBE-β-CD showed that Dap can have stronger interactionswith EPCLs than with cyclodextrins. The formation of complexesDap/β-CD and Dap/SBE-β-CD was further confirmed by comparingthe Dap longitudinal relaxation time (T1) in the absence andpresence of the cyclodextrins, revealing that Ha changed from2.84 to 1.81 s and Hb from 3.29 to 2.20 s in Dap/β-CD, confirmingthe formation of the inclusion complex. Analogous behaviourwas observed for both Ha and Hb in the absence and presence of

Figure 1. Representative Job’s plot for the complexation of (A) dapsone 1 anD2O with phosphate buffer at pH 7. The total concentrations were 2mmol l�

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SBE-β-CD, changing T1 from 2.84 to 1.59 s and from 3.29 to 1.85 s,respectively. The Dap/β-CD and Dap/SBE-β-CD stoichiometrieswere determined using the Job’s plot method,[29] which derivesfrom the NMR analyses. The concentration sum of Dap and β-CD(or SBE-β-CD) was maintained at 2mmol l�1, and the formation ofthe 1 : 1 complexes for Dap/β-CD and Dap/SBE-β-CD was observed,as described in the preceding texts for the Dap/β-CD complex[29]

(Fig. 1). The Job’s plot method has not been applied to EPCLs,and our own attempts failed to produce any understandable resultas the chemical shifts of the Dap hydrogens were not sensitive toEPC concentration.

The architectural arrangement of Dap/β-CD was proposedbased on detailed data provided by the 1D ROESY. Such resultshave already been presented in the preceding texts in differentexperimental conditions;[31,32] however, we repeated the

d β-CD 2 and (B) dapsone 1 and SBE-β-CD 3.The samples were prepared in1, and the 1H chemical shifts were measured at 298 K at 400.1315MHz.

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Dapsone in EPC liposome, SBE-β-CD and β-CD

experiments paying special attention to the selective 180° pulseused in the 1D ROESY and quantification by integrating theROE values to obtain a detailed structure of the complex. The sig-nal enhancements revealed dipolar cross relaxations between β-CD H3 and Dap Ha and Hb (0.64 and 0.34%, respectively) andbetween β-CD H5 and Dap Ha and Hb (1.79 and 0.90%, respec-tively). The structure of the Dap/β-CD complex was sensitive tothe NMR experimental parameters. Thus, in our experiments,Dap was fully inserted into β-CD, similar to some complexes stud-ied by 1H NMR[31,32] but different from others suggested basedon emission spectroscopy.[30] However, atomic level interactionsare specially observed by NMR spectroscopy, producing moreaccurate information regarding the architecture of the supra-molecular complexes. Figure 2 shows the 1D ROESY spectrumirradiating H3 of β-CD, and a symmetrical structure was proposedbased on the Dap Ha and Hb signal enhancements. SBE-β-CD 1HNMR signals were not well resolved, preventing any selectiveexcitation. Therefore, we focused on the enhancements occur-ring in regions and not at specific signal. Thus, cross relaxationswere detected between the Ha and SBE-β-CD signals at 5.0 to4.0 ppm (2.65% enhancement) and between Hb and SBE-β-CDsignals at 3.5 to 4.0 ppm (1.49% enhancement). Notwithstandingthe signal enhancements of the Dap/SBE-β-CD complex, a spatialarrangement was not suggested because of the lack of 1H NMRsignal dispersion.

The change in the Dap hydrogen chemical shifts in thepresence of liposomes indicated the formation of an inclusioncomplex, but ROESY could not reveal the Dap/EPC complexspatial arrangement because of liposome signal broadening.These characteristics are intrinsically associated with the assem-bling of discrete molecules of EPC and Dap into large supramo-lecular arrangements with small correlation times. Therefore,the complex Dap/EPC was investigated by an alternative meth-odology, i.e. STD NMR spectroscopy, which was successfully

Figure 2. 1D ROESY spectrum of the dapsone/β-CD complex at 298 K and 40β-CD H3 (3.90 ppm). Above, a structure suggested for the Dap/β-CD complex

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applied to study the local anaesthetic-liposome interactions.[21,34]

Self-assembling in liposomes is a known phenomenon thatcauses NMR spin diffusion, which is usually observed in largemolecules with long correlation times, such as proteins. Thus,saturation of a liposome NMR signal is intramolecularly andintermolecularly transferred by cross relaxation to the Dap signalsat the complex interface. Saturation at 0.5 ppm changed the Haand Hb intensities by 13% (Fig. 3), revealing the complex for-mation. Furthermore, all hydrogens of Dap displayed equalperturbation, which was interpreted as a full insertion of the druginto the lipid bilayer, with all hydrogens receiving the sameamount of magnetisation transfer from the liposome by crossrelaxation because the liposome and Dap do not have a specificinteraction site.

The architecture of binary and ternary complexes may influencethe morphology, stability and dimensions of the liposome, whichare important parameters to determine the drug loading capacity,release behaviour and circulation in vivo. Thus, the Dap/EPCcomplex is a promising binary system for drug delivery, and to im-prove its stability and drug loading capacity, β-CD was added tothe Dap–EPCL (complex Dap/EPC). Monitoring the ternary mixture(Dap, β-CD and EPC) with a 1D ROESY (Fig. 4) experiment, enhance-ments were observed when H3 β-CD was irradiated (Ha=1.70%and Hb=0.78%). However, H5 irradiation did not provoke anysignal enhancement, signalling that the architecture of Dap/β-CDin the ternary mixture had changed in the presence of the EPCLby partial removal of Dap from the β-CD cavity.

From the smaller and asymmetric magnetisation transfer(Hb has a greater enhancement compared with Ha, 0.94 and0.56%, respectively) of Dap in the STD NMR spectrum of Dap/β-CD/EPC system, it can be concluded that β-CD and EPCL arecompeting for Dap, thus decreasing the drug residence time inthe liposome vesicle (relative to the Dap/EPC system) and thesaturation degree. The presence of β-CD signals in the STD

0.1315MHz in D2O with phosphate buffer at pH 7. Selective irradiation ofin equilibrium with the uncomplexed dapsone and β-CD.

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Figure 3. 1H NMR spectra (400.1315MHz, 298 K, pH 7 and D2O/reference HDO 4.70 ppm) of dapsone in EPC liposomes (400 nm), (A) off-resonance STDspectrum (irradiating at 30 ppm) and (B) on-resonance STD spectrum (irradiating at �0.5 ppm). Above: dapsone structure and the relative degree ofsaturation transfer of the individual hydrogens and the proposed architecture for the Dap/EPC complex.

Figure 4. 1D ROESY spectrum of the dapsone/β-CD/EPC ternary com-plex at 298 K and 400.1315MHz in D2O with phosphate buffer at pH 7.β-CD H3 (3.83 ppm) selective irradiation.

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NMR spectrum of Dap/β-CD/EPC indicates that some saturationwas transferred to the β-CD, most likely via labile H exchangefrom the NH2 of Dap in the hydrophobic cavity of β-CD (Fig. 5).


Complexes Dap/β-CD, Dap/SBE-β-CD and Dap/EPC were alsoconfirmed by diffusion-ordered spectroscopy (DOSY NMR)experiments to provide additional data on these equilibria. Theirapparent association constants, Ka, were calculated from thediffusion coefficients of Dap, β-CD and SBE-β-CD determined inseparate experiments and in equilibrium with their respectivecomplexes. The Dap fraction complex previously defined for thecomplex Dap/β-CD, based on the diffusion coefficients obtainedby NMR, was 0.846,[31] whereas that obtained in this study was0.439. These differences were assigned to the experimentalprocedures; thus, the Dap/β-CD complex prepared from amixture of solutions containing Dap and β-CD separately and leftto reach equilibrium behaves differently from a complex preparedby coprecipitation and lyophilisation[31]. The latter induces theformation of a greater amount of complex as the solvent is slowlyreduced. The second hypothesis is based on the obstruction effectobserved in the diffusion coefficient measurements.[42–45] Diffusioncoefficients of guest molecules may vary because of the obstruc-tion effect caused by macromolecules and self-assembly, meaningthat such a phenomenon might be confused with the formation ofinclusion complexes. This effect is pronounced with an increasing


Figure 5. 1H NMR spectra (400.1315MHz, 298 K, pH 7 and D2O/reference HDO 4.70 ppm) of the ternary complex Dapsone/β-CD/EPC, (A) off-resonanceSTD spectrum and (B) on-resonance STD spectrum. Above, dapsone structure and the relative degree of saturation transfers of the individual hydrogensare shown.


concentration of molecules in the sample and can be monitoredby changes in the diffusion coefficient of the solvent or added tothe sample probe. This phenomenon was not taken into accountpreviously[31] but was considered in this work based on the varia-tion of the residual HDO diffusion coefficient, as seen in Fig. 6.Assuming that the obstruction effect was not significant and there-fore the changes in the diffusion coefficients are because of theformation of inclusion complexes, the apparent association con-stants, Ka, were calculated for all systems. The complex Dap/β-CDhas a Ka of 1396 lmol�1, whereas the Dap/SBE-β-CD complexhas a Ka of 246 lmol�1, indicating that Dap has greater affinity forthe β-CD cavity compared with SBE-β-CD (Table 1). The Dap/EPCcomplex apparent association constant of 84 lmol�1 was alsodetermined in the same manner (Table 1). This value is higher thanthose observed for local anaesthetics,[21,34] and taking into consid-eration that the apparent association constant was calculated usingthe EPC concentration and not the EPCL vesicles, this value could

Figure 6. Evaluation of the HDO and dapsone molecular diffusion coef-ficients D in different samples, measured at 298 K and 400.1315MHz withpH 7 phosphate buffer.


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be higher, indicating the high Dap affinity for the EPCL, which isconsistent with Dap solubility in this system and the observedchemical shift variations.

The ternary system Dap/β-CD/EPC was also characterised bycomparing the diffusion coefficients (Fig. 7; Table 1) of the ternarysystem and the free molecules. The Dap diffusion coefficientsdecreased from 5.24×10�10m2 s�1 (free), 3.87 × 10�10m2 s�1 and4.30× 10�10m2 s�1 (Dap/β-CD and Dap/EPC, respectively) to1.75× 10�10m2 s�1 (Dap/β-CD/EPC), a value close to the β-CDdiffusion coefficient, indicating that the molar fraction offree Dap in solution is rather small and can be neglected. Inother words, the fraction of Dap complexed with β-CD is app-roximately equal to 1 [for Dap in the ternary system Dobs = x Dfree +(1� x) Dcomp. x→ 0].Therefore, the ternary system could be rationalised as a

mixture of two entities, a Dap/β-CD complex and an EPCL, withan association constant of the Dap/β-CD complex to EPCLthat was calculated using the β-CD diffusion coefficient in theDap/β-CD system (2.14 × 10�10m2 s�1) compared with theDap/β-CD diffusion coefficient in the Dap/β-CD/EPC system(2.01 × 10�10m2 s�1). It is therefore evident that there is aformation of the ternary complex, which is in equilibrium with thebinary complex Dap/β-CD and free EPCL according to Eqn (1). Theapparent association constant for this complex is 18 lmol�1.

Dap-β-CD þ EPC⇌Dap-β-CD-EPC (1)

Based on the signal enhancement in the 1D ROESY and STDNMR experiments, we proposed a structure for the ternary com-plex in equilibrium with the binary complex, as shown in Fig. 8.

Experimental

Chemical and reagents

Dap 1 (98%) and SBE-β-CD 3were purchased fromNúcleo de Tecno-logia Farmacêutica, Universidade Federal do Piauí, and D2O (99.9%)was purchased from Cambridge Isotope Laboratories, Inc.®. β-CD 2


Table 1. Diffusion coefficients (D) for pure substances and complexes and HDO diffusion coefficients

Complex Compound D (10�10m2 s�1) D HDO (10�10m2 s�1) Complex molar fraction f Ka (lmol�1)

— Dap 5.24 ± 0.06 16.68± 0.09 — —

— β-CD 2.12 ± 0.01 16.27± 0.02 — —

— SBE-β-CD 1.68 ± 0.02 16.10± 0.03 — —

— EPC 0.75 ± 0.08 16.86± 0.20 — —

Dap in β-CD Dap 3.87 ± 0.03 16.37± 0.02 0.4391 1396

β-CD 2.14 ± 0.04 — — —

Dap in SBE-β-CD Dap 4.30 ± 0.06 17.07± 0.04 0.1694 246

SBE-β-CD 1.75 ± 0.08 — — —

Dap in EPC Dap 4.00 ± 0.06 16.83± 0.10 0.2831 84

EPC 0.86 ± 0.08 — — —

Dap/β-CD in EPCa Dap/β-CD 2.01 ± 0.06 — 0.0828 18

EPC 0.57 ± 0.05 — — —

aDap/β-CD diffusion coefficient calculation in the Dap/β-CD/EPC complex with regard to EPC.

Complex molar fraction DDap�Dcomplex

DDap�Dmacro moland apparent complex association constant Ka ¼ f

1�fð Þ macro molecule½ ��f Dap½ �ð Þ.

Figure 7. Representative 1H NMR DOSY experiment for the ternary complex (β-CD, Dap and EPC liposome 400 nm) at 298 K and 400.1315MHz in D2Owith phosphate buffer pH at 7.

Figure 8. Representative structure proposed for the Dap/β-CD/EPC ternary complex.

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(99%) and EPC 5 were purchased from Aldrich®. Tert-butyl-calix[4]arene 4 was synthesised in our laboratory according to methodsfound in the literature.[39]

Sample preparations

EPC liposome 400 nm

A film of EPC was prepared by evaporating the solvent of an EPCsolution in chloroform. The film was resuspended in 0.1mol l�1

phosphate/biphosphate buffer at pH 7, in D2O, and extrudedthrough a 400-nm polycarbonate membrane (micro-extruder,12 cycles). The total lipid concentration was 5mmol l�1.

Dapsone/β-CD and dapsone/SBE-β-CD complexes

The systems Dap/β-CD and Dap/SBE-β-CD were prepared bysolubilising Dap and each cyclodextrin separately in 700μl of0.1mol l�1 phosphate/biphosphate buffer at pH 7, in D2O, andthe binary complexes were gently stirred for 12 h. Dap finalconcentration was 1mmol l�1.

Dapsone/EPC complex

For the Dap–EPC system, Dap was slowly stirred for 4 h with amagnetic stirrer in 700μl of liposome suspension (400 nm, previ-ously prepared in D2O and 0.1mol l�1 phosphate/biphosphatebuffer at pH 7). The Dap final concentration was 1mmol l�1.

Dapsone/β-CD/EPC complex

The ternary complex was prepared by adding Dap/β-CD binarycomplex to 700μl of liposome suspension (400 nm, previouslyprepared in D2O and 0.1mol l�1 phosphate/biphosphate bufferat pH 7) to reach 1mmol l�1 of Dap and β-CD and 5mmol l�1 oflipid concentration.

NMR measurements

All NMR experiments were performed at 298 K in D2O and at pH 7on a Bruker Avance 400 spectrometer operating at 9.4 T. Thespectrometer was equipped with a 5-mm triple-resonanceinverse detection probe with Z-gradient (Probe PH TBI 600S3H/C-BB-D-05 Z). The 1H NMR chemical shifts are given in ppmrelative to the residual HDO signal at 4.7 ppm.

ROE measurements: The 1D ROESY experiments were obtained(selrogp.2 pulse sequence) with 2048 scans, 32 kpoints for eachscan, spinlock mixing of 0.5 s, repetition time of 5 s, acquisition timeof 2 s and exponential weighting of lb = 2. The 180°-shaped pulse(Gaus1.180r.1000) was set with 100ms and 1.73μW of power.

The diffusion experiments were conducted by ‘one-shot pulsesequence’ for high-resolution DOSY (100 scans, 32 kpoints foreach scan, acquisition time of 2 s and repetition time of 3 s).The 16 pulsed gradients ranged from 0.017 to 0.272 Tm�1,decreasing the signal intensity by 80–90% at the largest gradientamplitude, with a diffusion time of 0.06 s. The spectra wereprocessed by the DOSY toolbox programme[46] (32 kpoints forchemical shifts dimensions, 256 points for diffusion coefficientsdimension and using lb = 1 as the exponential weighting), fittingthe decay curve for each peak to an exponential decay andobtaining the diffusion coefficient and the pseudo two-dimensional spectra with NMR chemical shifts along one axisand calculating the diffusion coefficients (m2 s�1 × 10�10) alongthe other.

All STD experiments (performed by stddiffgp.19 pulsesequence) were selectively saturated using Gaussian train pulses


at �0.5 ppm (50- and 1-ms delay between the pulses and 0.1mWof power) for the on-resonance and 30 ppm for off-resonance(saturation time of 2 s, acquisition time of 2 s, repetition time of3 s, spinlock of 100ms, 2048 scans with 32 kpoints for each scanand lb = 3 as the exponential weighting).

Conclusions

Changes in the chemical shifts, diffusion coefficients and longitu-dinal relaxation times indicate the formation of the Dap/SBE-β-CDbinary complex; however, the lack of signal dispersions in the 1HNMR spectrum prevented determination of the complex architec-ture. The large variation in the chemical shift and the intensesignal observed in the STD experiment suggest a strong interac-tion between Dap and 400-nm EPCLs with full insertion of Dapinto the liposome lipid bilayer. The results of associated experi-ments, such as 1D ROESY, STD and diffusion coefficient measure-ments, indicated the formation of the strong ternary complexDap/β-CD/EPCL. This type of ternary system may be an alterna-tive application of the poorly soluble Dap. Additionally, we deter-mined the apparent association constants, Ka, for three binarycomplexes and one ternary complex based on variations in thevalues of the diffusion coefficients: Dap/β-CD (Ka= 1396 lmol�1),Dap/SBE-β-CD (Ka= 246 lmol�1), Dap/EPCL (Ka= 84 lmol�1) andDap/β-CD/EPCL (Ka= 18 lmol�1).

Acknowledgements

The authors are grateful to Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES)/Direção Geral de Universidades(CAPES no. 181/09), Conselho Nacional de DesenvolvimentoCientifico e Tecnologico, Fundacao de Amparo a Pesquisa doEstado de Sao Paulo and Petrobrás for financial support andscholarships. We are also indebted to Carol H. Collins (Universityof Campinas) for manuscript revision.

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