LPDT Novel Potential Drug Against T. cruzi and Its Interaction with Surfactant Micelles

Pharmaceutical Development and Technology, 12:183–192, 2007 Copyright © Informa HealthcareISSN: 1083-7450 print / 1097-9867 onlineDOI: 10.1080/10837450701212727

183

LPDT

Novel Potential Drug Against T. cruzi and Its Interaction with Surfactant Micelles

Potential Drug Against T. cruzi and Interaction with MicellesCarlota O. Rangel-YaguiDepartment of Biochemical and Pharmaceutical Technology, Faculty of Pharmacy, University of São Paulo, São Paulo, Brazil, and Department of Pharmacy, Faculty of Pharmacy, University of São Paulo, São Paulo, Brazil

Helen W.L. HsuDepartment of Biochemical and Pharmaceutical Technology, Faculty of Pharmacy, University of São Paulo, São Paulo, Brazil

Leandro R.S. Barbosa and Wilker CaetanoInstitute of Physics, University of São Paulo, São Paulo, Brazil

Adalberto Pessoa Jr and Leoberto C. TavaresDepartment of Biochemical and Pharmaceutical Technology, Faculty of Pharmacy, University of São Paulo, São Paulo, Brazil

Rosangela ItriInstitute of Physics, University of São Paulo, São Paulo, Brazil

The interaction of 5-nitro-2-furfurilylidene benzhydrazide(5NFB), potential anti-trypanosomal compound, with micellarsolutions was studied. The results indicated that 50 μg of 5NFBcompletely kills 20 million T. cruzi epimastigote cells within 3days, whereas the same amount of benznidazole kills 30% of thecells after 4 days. 5NFB solubility in surfactants solutions (SDS,DTAB, C12EO8) increased linearly with surfactant concentration.According to small angle X-ray scattering (SAXS), 5NFB doesnot affect micellar structural features. A comparison betweenC12EO8 effects on T. cruzi epimastigote cells and on erythrocytesshowed that surfactant lytic effect is stronger in parasite cells,enlightening the potential of 5NFB micellar formulations.

Keywords micelle-drug interaction, p-substituted benzhydrazides,anti-trypanosomal activity

INTRODUCTION

Chagas’ disease is an important social and medicalailment for people living in the Americas. It is endemic in21 countries, with approximately 16–18 million individu-als infected with Trypanosoma cruzi. Current therapy isbased on nifurtimox and benznidazole, which are mostlyeffective in the acute phase of the disease and may causeserious side effects. In Brazil, benznidazole is the onlydrug available, despite the claim that Brazilian T. cruzistrains are more resistant to this drug than strains fromother countries.[1] Therefore, the search for new and betterdrugs against T. cruzi is of paramount importance.

Despite their toxicity, nitro aromatic compounds aregenerally very active against T. cruzi and have been con-sidered as important leads for molecular modification.[2,3]

In this respect, the 5-nitro-2-furfurilidene benzhydrazide(5NFB) (Figure 1) is the lead of a series of nitro-heterocycliccompounds synthesized by Tavares et al.[4] that havealready proved to be active against multidrug-resistantStaphylococcus aureus and represent potential drugs anti-trypanosome. Nevertheless, the 5NFB water solubility isonly 20 μmol/L, and the logPo,w value was calculated to be1.51 (ClogP™, BioByte, Claremont, CA). The low solu-bility constitutes an important drawback in formulation,

Received June 2006, Accepted 25 October 2006.Address correspondence to Carlota O. Rangel-Yagui, Av. Prof.

Lineu Prestes, 580 – Bloco 15, CEP: 05508-950 – São Paulo, SP;E-mail: [email protected]

184 C.O. Rangel-Yagui et al.

and, in this sense, the use of surfactants could be an inter-esting alternative.

Surfactants are amphiphilic molecules consisting of ahydrophilic portion and a hydrophobic chain. The ambiva-lence of amphiphiles toward an aqueous environment isresponsible for the self-assembly of individual surfactantmolecules resulting in a variety of micellar aggregate struc-tures.[5] The concentration of surfactant molecules at whichmicelles appear is called critical micelle concentration(CMC). In a micelle, the hydrophobic tails flock to the inte-rior to minimize their contact with water, and the hydro-philic heads remain on the outer surface to maximize theircontact with water.[6] The micellization process in waterresults from a delicate balance of intermolecular forces,including hydrophobic, steric, electrostatic, hydrogenbonding, and van der Waals interactions. The main attrac-tive force results from the hydrophobic effect associatedwith the nonpolar surfactant tails, and the main opposingrepulsive force results from steric interactions and electro-static interactions between the surfactant polar heads.Whether micellization occurs and, if so, at what concentra-tion of monomeric surfactant, depends on the balance ofthe forces promoting micellization and those opposing it.[7]

Special attention has been paid to micelles in thepharmaceutical field owing to their ability to increase thesolubility of sparingly soluble substances in water.[8,9] Inthis context, solubilization can be defined as the spontane-ous dissolving of a substance by reversible interaction withthe micelles of a surfactant in water to form a thermodynam-ically stable isotropic solution with reduced thermodynamic

activity of the solubilized material.[10] The use of aqueousmicellar solutions for drug solubilization may also be advan-tageous for drug delivery purposes, with the possibility ofimproving bioavailability, reducing toxicity and other sideeffects, enhancing permeability across the physiological bar-riers, and substantially changing drug distribution.[11]

In this work, we evaluated the activity of 5NFBagainst T. cruzi and compared it with the reference drug forChagas’ disease benznidazole. Considering the low watersolubility, we also investigated the interaction of 5NFBwith nonionic and ionic micelles aiming at future directionsfor drug formulation. Three surfactants, a nonionic [n-dodecylocta(ethylene oxide)], a cationic (n- dodecyltrimethylam-monium bromide), and an anionic (sodium dodecyl sul-fate), all presenting the same tail length but different headgroups (Figure 1), were studied. Solubility measurementsof 5NFB in micellar solutions were performed, and thestructural features of the micelle-like aggregates wereinvestigated by means of small angle X-ray scattering(SAXS). In fact, SAXS has been quite often used as a pow-erful tool by some of us to study micelle shape transforma-tion induced by the interaction with some drugs such aslocal anesthetics,[12] phenothiazines,[13–15] and porphy-rins.[16] Although some surfactants, especially ionic ones,might be toxic and present restrictions for in vivo use, bystudying the solubilization of 5NFB in model micellar sys-tems we hope to contribute to the fundamental understandingof the micellar solubilization behavior of nitro-heterocycliccompounds. In future studies, we plan to investigate solubi-lization in polymeric micelles, more stable and promisingfrom a pharmaceutical perspective.

The hemolytic potential of the nonionic surfactantC12EO8 was measured to evaluate the use and possibleadvantages of micellar formulations of 5-nitro-2-furfurilidenecompounds to treat Chagas’ disease. We have not measuredhemolytic potentials of the other surfactants studied becauseof their stronger restrictions for in vivo use.

MATERIALS AND METHODS

Materials

The cationic surfactant n-dodecyltrimethylammoniumbromide (DTAB) was from Sigma (St. Louis, MO). Theanionic surfactant sodium dodecyl sulfate (SDS) was fromPharmacia Biotech (Uppsala, Sweden). The nonionic sur-factant n-dodecyl octa(ethylene oxide) (C12EO8) was fromNikko Chemicals (Tokyo, Japan). The 5-nitro-2-furfurilidenebenzhydrazide (5NFB) synthesized was readily availableat the Laboratory of Drug Planning and Development,School of Pharmacy, University of São Paulo, under thesupervision of Prof. Leoberto Costa Tavares. All the other

Figure 1. Chemical structure of the compound 5-nitro-2-furfurylidene benzhydrazide (5NFB, I) and the surfactants:anionic sodium dodecyl sulfate (SDS – II), cationicdodecyltrimethylammonium bromide (DTAB – III), andnonionic dodecyl octa (ethylene oxide) (C12EO8 – IV).

O

NN

ONO2

(OCH2O)8

N CH3

CH3

CH3+

–Br

II

III

IV

I

O S

O

O

+Na– O

Potential Drug Against T. cruzi and Interaction with Micelles 185

reagents were of analytical grade. The solutions were pre-pared in 5 mmol/L phosphate buffer at pH 7.4 (ionicstrength = 0.011 mol/L) by using water purified through aMillipore Milli-Q ion-exchange system (Bedford, MA).

In Vitro Determinations of Activity Against T. cruzi

The epimastigote culture of T. cruzi, Y strain, waskept in liver infusion tryptose (LIT) medium composed of0.4% NaCl, 0.04% KCl, 0.8% Na2HPO4, 0.2% glucose,0.5% tryptose, 0.5% liver infusion (all w/v percentages);enriched with 10% (v/v) bovine serum. The medium wasadjusted to pH = 7.2 and incubated at 68°C during 1 hr.After cooling, a bovine hemoglobin solution was added at2% (v/v). The medium was sterilized by filtration througha 0.22-μm membrane (Millipore) and stored at 4°C.

The systems were prepared in LIT medium with an inoc-ulum of 20 × 106 parasites/mL (obtained from 71-hr cultures)and known concentrations of the compound to be tested(5NFB, benznidazole, or C12EO8). If necessary, up to 3% ofdimethylsulfoxide was added to allow solubilization of thedrugs. A control consisting only of inoculum in LIT and thesame concentration of DMSO used in the experiments withthe compound was included to check the parasite growth.Then the systems were incubated at 28°C and the trypanomi-cidal activity was measured by means of counting the surviv-ing epimastigotes in a Neubauer camera after 0, 1, 2, 3, and 4days. All the experiments were carried out in triplicate.

Determination of C12EO8 Hemolytic Potential

The systems were prepared in sodium phosphatephysiological buffered solution (PBS) having 5% (v/v) ofsheep blood and increasing concentrations of C12EO8. Thesystems were then kept in a water bath at 37°C for 1 hr,under agitation, followed by centrifugation at 1400 rpmfor 5 min. Finally, the supernatant absorbance at 540 nmwas measured (Asample) and compared to the absorbance ofa system prepared with water (Awater) to give the percent-age of hemolysis, according to Eq. (1). All the experi-ments were carried out in triplicate.

Determinations of Critical Micelle Concentrations (CMC)

The determination of a surfactant CMC can be made byuse of several physical properties, such as surface tension,

conductivity in ionic surfactants, osmotic pressure, deter-gency, etc. When these properties are plotted as a functionof surfactant concentration (or its logarithm, in surface ten-sion), a sharp break can be observed in the curves obtained,evidencing the formation of micelles at that point.[17]

The CMC of the surfactants at 25°C was determinedin pure water, phosphate buffer pH 7.4, as well as in phos-phate buffer in the presence of 5NFB at saturation (20μmol/L). The CMC determinations for SDS and DTAB werebased on the change in conductance with surfactant concen-tration by using a MPC 227 Mettler-Toledo conductivimeter(Columbus, OH). The CMC determinations for C12EO8 werebased on the change in surface tension with surfactant con-centration. A DuNoüy ring tensiometer (TE 1C/3 Lauda-Königshefen, Germany) was used, and the effect of curvaturewas accounted for with the Harkins and Jordan correctionfactor.[18] Each conductivity/surface tension measurementwas repeated three times, and the typical error in the CMCdetermination was less than 5%.

Solubility Studies

The solubility of 5NFB in SDS, DTAB, and C12EO8aqueous solutions was measured at surfactant concentra-tions between 0 and 100 mmol/L. Excess amounts of5NFB were added to vials containing 2.0 mL of bufferedsurfactant solutions (SDS, DTAB, or C12EO8). The samplevials were then rotated at 8 rpm in an end-to-end rotator(Labquake, Barnstead/Thermolyne, Dubuque, IW) at 25°Cfor 24 hr. According to previous experiments, 24 hr wasenough for the system to reach solubility equilibrium (datanot shown). After this period, the samples were filteredthrough 0.20-μm Minisart RC 25 filters (Sartorius,Goettingen, Germany), and the concentration of solubi-lized 5NFB was determined spectrophotometrically at l =376 nm in a Beckman DU 640 spectrophotometer (Fuller-ton, CA), with the help of a calibration curve. For all theabsorbance measurements, surfactant solutions at the sameconcentration of the samples were used as blanks. All thesolubility experiments were carried out in triplicate.

The molar solubilization capacity, c, was calcu-lated on the basis of the general equation for micellarsolubilization[19]:

where Stot is the total drug solubility, SW is the drug solu-bility in phosphate buffer, Cs,mic is the molar surfactantconcentration in the micellar form that can be approxi-mated as (Cs–CMC), with Cs corresponding to the molarconcentration of surfactant in solution. Therefore, c is theratio of drug concentration in the micelles to the surfactant

%hemolysisA

Asample

water

=(1) S S Ctot W s mic= + χ , (2)


concentration in the micellar form and corresponds to theangular coefficient of the linear regression of a Stot versusCs,mic plot.

Micelle-water partition coefficients (K) were calcu-lated for surfactant concentrations of 40 and 80 mmol/L,based on the following equation[19]:

where, according to Eqs. (1) and (2):

Small Angle X-Ray Scattering (SAXS) Studies

SAXS experiments were performed at the NationalLaboratory of Synchrotron Light (LNLS, Campinas, Brazil)at 22°C, with radiation wavelength l = 1.608 Å and sam-ple to detector distance of about 720 mm. Isotropic solu-tions of the surfactants SDS, DTAB, and C12EO8 (40 and80 mmol/L) in phosphate buffer in the absence and pres-ence of 5NFB (100 and 200 μmol/L) were investigated.Samples were conditioned in a 1-mm-thick Mylar cell,positioned perpendicular to the incident X-ray beam. Theobtained curves (data collection of 15 min) were correctedfor detector homogeneity (one-dimensional position-sensitivedetector) and normalized by taking into account thedecrease of the X-ray beam intensity during the experi-ment. The parasitic background (buffer solution) was sub-tracted considering the sample’s attenuation.

SAXS Theory

The observed scattering intensity [I(q)] of an isotropicsolution of monodisperse spheroid particles of low anisot-ropy can be described as[20]:

where np corresponds to the particle number density, k is anormalization factor related to the instrumental effects,P(q) is the single particle form factor, reflecting the micellesize and shape, S(q) is the interparticle structure factor, andq (=4psinq/l, 2q is the scattering angle) is the modulus ofthe scattering vector. For systems with small polydispersity(∼20%), the deviation in Eq. (4) corresponds to a diffusescattering that was accounted for in our data treatment.[21]

In the present work, we have used small prolate ellip-soids for micelle shape:[21,22] the shortest semi-axis is onthe order of the paraffinic chain length, Rpar (the firstmodel parameter), whereas the largest semi-axis is vRpar,with v equal to the axial ratio (the second parameter). Themicelle is constituted by two well-defined shells of differ-ent electron densities: an inner core of paraffinic moiety,with electron density rpar = 0.275 e/Å3, and an externalshell, surrounding the core, with a respective polar headgroup thickness σ that includes the hydration water, andelectron density ρpol (third and fourth parameters) relativeto the continuous medium (buffer solution with electrondensity assumed to be similar to that of the water ρw =0.333 e/Å3). A more detailed discussion regarding themodel can be found in previous publications.[13,14,21]

S(q) in Eq. (1), as mentioned above, is the interparti-cle structure factor. In this work, we made use of the well-known mean spherical approximation (MSA), developedby Hayter and Penfold.[23,24] This method approximatesthe scattering particles as charged spheres with ionizationcoefficient α = z/n (where z and n are, respectively, themicelle charge and the aggregation number) interactingthrough a screened Coulomb potential. Noteworthy, theuse of α, as a free parameter, can suppress some deficien-cies of the adopted model potential. In this regard, α valuecannot be directly related to the surface charge, but it hasto be interpreted as an effective parameter.[21]

The structural parameters are then obtained by fittingEq. (4) to the experimental data. The determination of k inEq. (4) is performed by considering the scattering intensityfrom a standard sample, once the respective P(q) and S(q)functions are well known.[13,14,16,21]

RESULTS AND DISCUSSION

The CMC results for the three surfactants studied aresummarized in Table 1. The values obtained in water are

KS S

Stot W

W

=−( )

(3)

KC

Ss mic

W

=χ , (4)

I q kn P q S qp( ) ( ) ( )= (5)

Table 1 Critical micelle concentrations (CMC) experimentally

determined for the surfactants SDS, DTAB, and C12EO8 in water, phosphate buffer, pH 7.4, and 5NFB-saturated (20 μmol/L)

phosphate buffer at pH 7.4

Critical micelle concentration–CMC (mmol/L)

Surfactant H2O Buffer Buffer + 5NFB

SDS 8.7(4) 4.7(2) 4.6(2)DTAB 15.9(6) 13.5(5) 13.6(5)C12EO8 0.081(4) 0.085(4) 0.076(4)

The numbers in parentheses correspond to the error associatedto the last significative number.


in agreement with previous values reported in literature,according to which CMCSDS = 8.2 mmol/L, CMCDTAB =16.0 mmol/L,[10] and CMCC12EO8 = 0.088 mmol/L.[25] TheCMCs of SDS and DTAB were depressed in phosphatebuffer mainly because of the decrease in the thickness ofthe electric double layer surrounding the ionic head groupsand decreased electrical repulsion between the headgroups in the micelle.[26]

According to Table 1, no statistically significant dif-ferences were observed in the CMC of the three surfac-tants studied in the presence of 5NFB, in comparison tothe buffer solutions. It has been described that moleculessolubilized in the outer portion of the micelle core aremost effective in reducing the CMC than the ones solubi-lized in the inner core.[10] Therefore, if solubilization of5NFB is taking place, the drug should be located in theinner core of the micelles, as a result of hydrophobic inter-actions with the surfactant tail.

Figure 2 presents solubility results for 5NFB as afunction of the concentration of surfactants: SDS (Figure 3a),DTAB (Figure 3b), and C12EO8 (Figure 3c). As expectedbased on previous works,[27] irrespective of the surfactanttype, the solubility of 5NFB increased linearly withincreasing surfactant concentration, as a consequence ofthe association between the compound and the micelles.The increase in drug solubility was observed only for sur-factant concentrations above the CMC. This finding can-not be visualized from the solubilization curves because

we have just presented solubility data for surfactant con-centrations above the CMCs. However, measurements atsurfactants concentrations below the CMC (data not pre-sented) have shown no change in 5NFB solubility, clearlydemonstrating that micellar solubilization is taking place.

According to the solubility determinations, the 80mmol/L SDS micellar solution resulted in a 10-foldincrease in 5NFB solubility compared to the buffer solu-tion, whereas the 80 mmol/L DTAB solution resulted in an8-fold increase, and 80 mmol/L C12EO8 solution in a 6-fold increase. Usually, nonionic surfactants are better solu-bilizing agents than ionic surfactants in dilute solutions,because of their lower CMC. Therefore, in C12EO8 micel-lar solutions, the solubility of the drug is not only a conse-quence of micelle-drug interaction but also of the fractionof surfactant in the micellar form. Nevertheless, the solu-bility of 5NFB was lower in C12EO8 than in SDS andDTAB micellar solutions. Krishna and Flanagan,[28] study-ing the antimalarial drug b-Arteether (an endoperoxidecontaining a sesquiterpene lactone), also observed lowersolubilization power of nonionic surfactants than ionicsurfactants. They suggested that the solubilization of this

Figure 2. Solubility curve of 5-nitro-2-furfurilydene benzh-ydrazide (5NFB) as a function of the surfactants: (a) sodiumdodecyl sulphate (SDS, -�-); (b) dodecyltrimethylammoniumbromide (DTAB, -Δ-); and (c) n-dodecyl octa (ethylene oxide)(C12EO8, -❍-). All the samples were prepared in phosphatebuffer (5 mmol/L), pH 7.4, at 25°C. The black diamond (�)represents 5NFB solubility in buffer. The error bars represent95% confidence limit for the measurements.

0

0.1

0.2

0.3

Surfactant Concentration - Csurf (mM)

5NFB

Sol

ubili

ty -

Sto

t (m

M)

c

b

a

0 10080604020

Figure 3. SAXS curves from the micellar solutions at 80mmol/L in the absence and presence of 5NFB, in phosphatebuffer solutions at pH 7.4 and room temperature of 22°C. Thethick lines represent the best modeling curves to the experimentaldata (❍), based on Eq. (5).

q (Å–1)

Inte

nsity

(a.u

.)0

1

0

1

2

3

0

1

2

3

DTAB

SDS

C12EO8

0.00 0.300.250.200.150.100.05

0.00 0.300.250.200.150.100.05

0.00 0.300.250.200.150.100.05


drug may not only involve incorporation into the micellarinterior but may be substantially due to adsorption at themicelle-water interface.

Mukerjee and Cardinal[29] studied the microenviron-ments of benzene, some of its derivatives, Triton X-100,and naphthalene when solubilized in micelles at lowsolubilizate to surfactant ratios and proposed the existenceof at least two states (loci) of solubilization with differentpolarity. According to the authors, the total uptake bymicelles could be divided approximately into an“adsorbed” fraction (location at the micelle-water inter-face) and a “dissolved” fraction (location in the hydrocar-bon core). When adsorption takes place, the solubilityincreases beyond the solubility power of the hydrocarboncore. In fact, numerous studies indicate that the solubilityof slightly polar substances and aromatic compounds tendto be considerably higher than the solubility of aliphaticcompounds presenting similar molar volumes, despite thefact that the latter are expected to be more compatible withthe aliphatic hydrocarbon core of most micelles.

However, for the hydrophobic compound 5NFB itseems more reasonable that most of it is solubilized in themicellar core. Solubility experiments of 5NFB in C16EO8aqueous micellar solutions carried out by our group (datanot shown) have resulted in a 1.3-fold increase in the com-pound solubility compared to C12EO8 at the same concen-tration, indicating 5NFB solubilization preferentially inthe inner core of the micelles. Nevertheless, although thethree surfactants studied present the same tail length,micellar self-assembly might be different and result in dif-ferences on the volume of the micellar core and, conse-quently, on the extent of solubilization.

The molar solubilization capacities of the surfactants,as well as the partition coefficients, were calculated andare presented in Table 2. According to the obtained values,

the molar solubilization capacity of SDS, c = 2.9 mmol/molis higher than the molar solubilization capacity of DTAB,c = 2.4 mmol/mol, and the lowest one is the molar solubi-lization capacity of C12EO8, c = 1.9 mmol/mol. In addi-tion, the partition coefficients results show that thetendency of 5NFB to partition preferentially to SDSmicelles is higher than the tendency to partition to DTABand C12EO8 micelles. SAXS experiments were performedon these systems to investigate if the 5NFB could impacton the micellar self-assembling features. For the drugstetracaine,[12] phenothiazines,[13–15] and porphyrins,[16]

micelle shape transformation was previously observed byadding up to 10 mmol/L of drug to 40 mmol/L of surfac-tant. It should be remarked that the solubility values for5NFB in the micellar systems studied are very low in com-parison to the solubility observed for these other drugs.

Figure 3 shows typical SAXS curves obtained fromall studied surfactants at 80 mmol/L in buffer solution atpH 7.4 in the absence of drug, which are identical to thoseobserved in the presence of up to 200 μmol/L of 5NFB.Therefore, the 5NFB aggregation within the micelles didnot alter the aggregates morphology as seen by SAXS. Allcurves obtained present a broad peak, around q ≈ 0.14 Å−1

in DTAB and C12EO8 curves and around 0.17 Å−1 in theSDS scattering curve. This peak is characteristic of theintramicellar form factor. On the other hand, the pres-ence of a second peak around q ≈ 0.04 Å−1 in the SDScurve clearly reveals the interaction between chargedmicelles.[13,21]

Table 3 presents the best fitting parameters for theSAXS measurements. Accordingly, the data from C12EO8micelles are consistent with quasi-spherical micelles [axialratio equal to 1.2(1)], with total radius around 29 Å (Rpar =22Å and σ ≈ 7 Å). There is a voluminous amount ofresearch on the structural features of nonionic surfactants.

Table 2 Experimentally determined solubility parameters for 5NFB in

aqueous surfactant solutions of SDS, DTAB, and C12EO8 (phosphate buffer at pH 7.4)

Surfactant Linear fit K40 K80 c (mmol/mol)

SDS y = 0.0031x + 0.006R2 = 0.990

5.5(3) 10.9(3) 2.9(3)

DTAB y = 0.0024x + 0.003 R2 = 0.995

3.9(4) 8.5(4) 2.4(4)

C12EO8 y = 0.0019x + 0.025 R2 = 0.994

3.8(2) 7.9(2) 1.9(2)

K40 and K80 are the 5NFB partition coefficients at 40 mmol/Land 80 mmol/L surfactant concentrations, respectively, and c isthe molar solubilization capacity. The numbers in parentheses cor-respond to the error associated to the last significative number.

Table 3 Adjustment parameters obtained from the scattering curves of the

surfactants SDS, DTAB, and C12EO8, 80 mmol/L micellar solutions in phosphate buffer at pH 7.4, through P(q) and S(q)

modeling [Eq. (4)]

Surfactant α Rpar (Å) σ (Å) ρpol (e/Å3) ν

SDS 0.14(1) 17(1) 6.2(4) 0.41(1) 1.6(1)DTAB 0.17(2) 17(1) 3.3(2) 0.41(1) 1.6(1)C12EO8 — 22(1) 7.3(9) 0.38(1) 1.2(1)

α is the degree of ionization at the surface of the micelle, Rpar

= the shortest paraffinic axis, σ = the polar shell thickness, ρpol =the polar shell electron density, and ν = the axial ratio betweenthe largest and the shortest paraffinic axis.

The numbers in parentheses correspond to the error associatedto the last significative number.


Magid et al.[30] studied the influence of temperature in thephase transition of surfactants of the type CmEOn. Theyalso evidenced, by means of small-angle neutron scatter-ing (SANS), the occurrence of nearly spherical micelleswith total radius around 27.1(3) Å (at 20°C), in agreementwith our results. Nilsson et al.[31] studied the structure ofnonionic micelles (C12EO8 and C12EO5) by nuclear mag-netic resonance self-diffusion and proton relaxation andalso observed small spherical micelles. These authors esti-mated micellar radius by extrapolating the micellar diffu-sion coefficients to infinite dilution approximation. Bydoing so, they obtained a radius of 31 Å at 25.2°C, alsocorroborating with our results.

It is of interest that according to our SAXS data analy-sis from C12EO8 system, the Rpar = 22 Å (Table 3) wassomewhat higher than the extended length of the alkylchain expected for a dodecyl chain according to Tanford’sformula: 16.7 Å.[6] We assign this difference to a not clearseparation between the hydrocarbon core and the hydro-philic polar region. Probably, our Rpar value takes intoaccount some of the ethylene units of the head group.

In terms of SDS micelles, the data are in good agree-ment with previous results,[12–14,21] and SDS micelles canbe well represented as small prolate ellipsoids of axialratio v = 1.6 and the shortest paraffinic axis Rpar = 17 Å,consistent to the extended length of a dodecyl chain.[6] Inaddition, the ionization coefficient a = 0.14 here deter-mined for SDS micelles at 5 mmol/L phosphate buffer isin agreement with values previously reported for SDS inpure water, a = 0.22,[21] and for SDS in 20 mmol/L phos-phate buffer, a = 0.11.[13] The reduction in the value of αin buffer is expected because of partial surface chargescreening by salt addition to water solution.

Regarding DTAB micelles, the data analysis also indi-cates the presence of small prolate ellipsoids of axial ratio v≈ 1.6 with shortest paraffinic axis Rpar = 17 Å, in goodagreement with previous SANS data.[32] Note that, in con-trast to what is seen for SDS micelles, no interference peakwas observed in the SAXS curve of DTAB solutions(Figure 4). Nevertheless, previous SANS data clearly showedsuch influence at q ≈ 0.075 Å−1[32] and, therefore, S(q) func-tion has also been accounted for in the SAXS data analysis,resulting in an ionization coefficient α = 0.17, in good agree-ment with the SANS data. The absence of an interferencepeak on DTAB SAXS curve results from the fact that themaximum of S(q) coincides with the first minimum of P(q). Itis noteworthy that other P(q) models could be used, such asthe dry-shell model reported for DTAB,[33,34] but the generalconclusions would hardly change.

As can be seen from the SAXS curves, although solu-bility experiments showed interaction between 5NFB andthe three surfactants studied, no changes in theamphiphiles self-assembling properties were noticeable

because this would result in distinct micelle morphologies.Perhaps, because the technique needs macroscopic changesin the structure of micelles, increase in drug concentrationmight be necessary. Particularly, significant ellipsoid tocylinder micelle shape transformation was recentlyobserved for SDS:phenotiazine at 4:1 molar ratio.[13] In thepresent work, the ratio between surfactant and drug in solu-tion is 350:1. Therefore, although interaction between thedrug and the surfactant indeed took place, it did not induceany cooperative effect on micellar aggregates.

Table 4 presents the results of anti-trypanosomalactivity for the compounds studied and the surfactantC12EO8. As can be seen, 50 μg of 5NFB completely killeda culture of 20 million epimastigotes cells of T. cruzi (Ystrain) within 3 days, with about 90% reduction in just 2days. On the other hand, the same amount of benznidazolekilled about 30% of the cells only after 4 days. For thisdrug, complete destruction of the parasite culture wasobserved after 12 days (data not shown). It is worthy tomention the therapeutic dose of benznidazole, which is 5–7 mg/kg/day.[35] According to results in Table 4, 5NFB isabout 5 times more potent than benznidazole in vitro.Therefore, one could envision a therapeutic dose of 5NFBaround 1 mg/kg/day. Of course ADMET (absorption, dis-tribution, metabolism, excretion, and toxicity) studies areimperative before any dose assumption is made. Neverthe-less, these activity results show the potential of 5NFB, aswell as of the entire series of p-substituted benzhy-drazides, as new agents to treat Chagas’ disease.

Regarding the effect of the surfactant on the parasite,100 μmol/L of C12EO8 immediately killed the 20 millionepimastigotes culture. In this respect, many pharmacologi-cally active compounds are amphiphilic or hydrophobicmolecules, which may undergo different kinds of association,

Figure 4. Curve of hemolytic effect (%) as a function ofC12EO8 concentration. Csat refers to the surfactant concentrationrequired to the onset of hemolysis (saturation), and Csol refers tothe surfactant concentration that leads to 100% hemolysis(membrane solubilization).

0

20

40

60

80

100

C12EO8 Concentration (μ mol/L)

% H

emol

ysis

Csat

Csol

0 500400300200100


and whose site of action in the organism frequently is theplasma membrane. Even if their target is intracellular, theinteraction with this first barrier plays a fundamental role.Several surface active drugs are reported to self-associateand bind to membranes, causing disruption and solubiliza-tion in a surfactant-like manner. In view of their ability toalter the permeability of cell, surfactants display antibacte-rial properties by acting on bacterial cell walls.[36] There-fore, our results indicate an effect of C12EO8 on theparasite cell membrane leading to death. Considering theeffects of both 5NFB and C12EO8 on the T. cruzi, one canenvision micellar formulations of 5NFB as powerfulweapons to combat this parasite. In vivo, these micellescould act on the parasite membrane and simultaneouslypromote solubilization of the poorly water-soluble 5NFB.Nevertheless, one should keep in mind the toxicity associ-ated to surfactants in drug formulations.

As a preliminary evaluation of C12EO8 toxicity, thehemolytic potential of this surfactant was determined. Ingeneral, the hemolytic test in vitro serves as a screeningmethod for the toxicity of lytic agents contained in intrave-nous formulations. Using this kind of test, the erythrocytedamage induced by these agents in vivo can be esti-mated.[37] Figure 4 presents the results of hemolytic poten-tial of the surfactant C12EO8. Arrows indicate thesurfactant concentration required to induce saturation (theonset of hemolysis), Csat, and total membrane solubiliza-tion (100% hemolysis), Csol.[38] We obtained a similar pro-file to the ones previously described in literature fornonionic surfactants.[25] Hemolysis is the disruption of thered blood cells and can be caused by the interaction ofchemical compounds with the cell membrane. The effectof surfactants on the stability of erythrocyte membranestends to be biphasic; at small surfactant/lipid molar ratios,they protect erythrocytes against hypotonic lysis, whereas

at high molar ratios, they induce hemolysis.[36] Thehemolytic process induced by surfactants can be describedas a bilayer-to-micelle transition. At low surfactant/lipidratios, the lamellar structure of the membrane is main-tained; at high ratios, mixed micelles of surfactant andmembrane phospholipids are formed.[25]

Despite the hemolytic potential of C12EO8, it is wellknown that many formulations are marketed by using highconcentrations of organic solvents and are usually, but notalways, diluted prior to injection. For intravenous injec-tion, for example, the amount of organic solvent adminis-tered is up to 20% ethanol (bolus) and 6% propyleneglycol (infusion). Nevertheless, concentrations of 21%ethanol and 5.7% propylene glycol are enough to inducehemolysis in 50% of healthy red blood cells.[39]

It is of interest that the lytic effect of the surfactant seemsto be stronger in the parasite cells than in erythrocytes. Weobserved that 100 μmol/L C12EO8 resulted in complete lysisof the 20 × 106 epimastigotes/mL culture but did not presentsignificant hemolytic effect, with the concentration of eryth-rocytes in the tests estimated to be 25 × 107 erythrocytes/mL.[40] This result corroborates the potential of micellar for-mulations of p-substituted benzhydrazides as medicinal alter-natives for Chagas’ disease. Needless to say, more in depthtoxicological and formulation studies are necessary to betterevaluate the potential of the 5NFB/C12EO8 combination, aswell as of other surfactant/p-substituted benzhydrazides com-binations. One possibility worth investigating is the use ofblock-copolymers that should combine better solubilizationcapacity with lower hemolytic potential.

CONCLUSION

In this work, we have shown the potential of a novelcompound, 5-nitro-2-furfurilydene benzhydrazide (5NFB),representing a series of promising drugs against T. cruzi.This compound was proven to be 5 times more effectivethan the drug of choice for Chagas’ disease, benznidazole.Because of low aqueous solubility, micellar solubilizationof 5NFB was also investigated in SDS, DTAB, and C12EO8solutions. Despite the interaction of the drug with micelles,shown by the partition coefficient values obtained, therewere no changes in the morphological features of themicelles for the three surfactants studied, as evidenced bySAXS. The very low solubility, in the micro molar range,probably made it difficult to promote differences in micellarstructural features. Nevertheless, micellar solutions haveshown to be useful to increase the solubility of 5NFB. Inaddition, a strong lytic effect of the nonionic surfactantC12EO8 in the parasite cells was observed, making micellarformulations of the p-substituted benzhydrazides even moreinteresting alternatives for the treatment of Chagas’ disease.

Table 4 Experimentally determined activity of 5NFB, benznidazole, and

C12EO8 against epimastigote cells of T. cruzi, Y strain

Number of epimastigotes (expressed in millions)

DaysGrowth control

5NFB 50 μg/mL

Benznidazole 50 μg/mL

C12EO8 50 μg/mL

(100 μmol/L)

0 21 22 20 01 32 12 22 —2 50 2 23 —3 62 0 16 —4 61 — 14 —

The results correspond to the average number of cells afterexposition to each compound at the given concentrations. Theexperimental error in the determinations was about 10%.


ACKNOWLEDGMENTS

The authors thank the National Laboratory of Synchro-tron Light (LNLS, Campinas, Brazil) for the use of its facili-ties. This work was supported by research grants andfellowships from FAPESP (Fundação de Amparo à Pesquisano Estado de São Paulo-Brazil), CAPES (Coordenação deAperfeiçoamento de Pessoal de Nível Superior-Brazil), andCNPq (Conselho Nacional de Desenvolvimento Científico eTecnológico-Brazil). We also thank Prof. Omar A. M. A. ElSeoud for the use of the tensiometer and Prof. Maria JúliaManso Alves and MSc. Marinei F. Gonçalves for providingthe LIT medium for the biological activity determinations.

REFERENCES

1. Chung, M.C.; Guido, R.V.C.; Martinelli, T.F.; Goncalves,M.F.; Polli, M.C.; Botelho, K.C.A.; Varanda, E.A.; Colli,W.; Miranda, M.T.M.; Ferreira, E.I. Synthesis and in vitroevaluation of potential antichagasic hydroxymethyinitrofura-zone (NFOH-121): A new nitrofurazone prodrug. Bioorg.Med. Chem. 2003, 11 (22), 4779–4783.

2. Cerecetto, H.; Gonzalez, M. Chemotherapy of Chagas’ dis-ease: Status and new developments. Curr. Top. Med. Chem.2002, 2, 1187–1213.

3. Maya, J.D.; Bollo, S.; Nunes-Vergara, L.J.; Squella, J.A.;Repetto, Y.; Morello, A.; Perie, J. Chauviere, G. Trypanosomacruzi: Effect and mode of action of nitroimidazole and nitrofu-ran derivatives. Biochem. Pharmacol. 2003, 65, 999–1006.

4. Masunari, A.; Rezende, P.; Tavares, L.C. QSAR studies ofnifuroxazide analogs with antimicrobial activity against mul-tidrug-resistant Staphylococcus aureus. In 15th EuropeanSymposium on Quantitative Structure-Activity Relationships &Molecular Modeling, Istanbul. Program & Abstract, 2004, 123.

5. Blankschtein, D.; Thurston, G.M.; Benedek, G.B. Phenome-nological theory of equilibrium thermodynamic propertiesand phase separation of micellar solutions, J. Chem. Phys.1986, 85, 7268–7288.

6. Tanford, C. The hydrophobic effect: Formation of micellesand biological membranes. Wiley: New York, 1980.

7. Israelachvili, J.N. Intermolecular and Surface Forces, 2nded.; Academic Press: London, 1991.

8. Mall, S.; Buckton, G.; Rawlins, D.A. Dissolution behaviourof sulphonamides into sodium dodecyl sulphate micelles: Athermodynamic approach. J. Pharm. Sci. 1996, 85, 75–78.

9. Rangel-Yagui, C.O.; Pessoa-Jr, A.; Tavares, L.C. Micellar sol-ubilization of drugs. J. Pharm. Pharma. Sci. 2005, 8, 147–163.

10. Rosen, M.J. Surfactants and interfacial phenomena, 2nd ed.;John Wiley & Sons: New York, 1989.

11. Torchilin, V.P. Structure and design of polymeric surfactant-basedrug delivery systems. J. Control. Release 2001, 73, 137–172.

12. Teixeira, C.V.; Itri, R.; Casallanovo, F.; Schreier, S. Local anes-thetic-induced microscopic effects in micelles. A fluorescence,spin label and SAXS study. Biochim. Biophys. Acta 2001,1510, 93–105.

13. Caetano, W.; Gelamo, E.; Tabak, M.; Itri, R. Chlorprom-azine and sodium dodecyl sulfate mixed micelles investi-gated by small angle x-ray scattering. J. Coll. Inter. Sci.2002, 248, 149–157.

14. Caetano, W.; Barbosa, L.R.S.; Itri, R.; Tabak, M. Trifluopera-zine effects on anionic and zwitterionic micelles: A study bysmall angle X-ray scattering. J. Colloid Int. Sci. 2003, 260,414–422.

15. Barbosa, L.R.S.; Caetano, W.; Santiago, P.; Melo, P.H.; Tabak,M.; Itri, R. Phenothiazine Compounds Interaction with Zwitte-rionic Lysophosphatidilcholine LPC Micelles: Small AngleX-ray Scattering. The Journal of Physical Chemistry B,2006, 110, 13086–13093.

16. Gandini, S.C.M.; Itri R.; Neto, D.S.; Tabak, M. Porphy-rins effects on zwitterionic HPS micelles as investigatedby small angle x-ray scattering (SAXS) and electron para-magnetic resonance (EPR). J. Phys. Chem. B 2005, 109,22264–22272.

17. Jones, M.N.; Chapman, D. Micelles, Monolayers andBiomembranes, Wiley-Liss: New York, 1995.

18. Theander, K.; Pugh, R.J. The influence of pH and tempera-ture on the equilibrium and dynamic surface tension of aque-ous solutions of sodium oleate. J. Colloid Interf. Sci. 2001,239, 209–216.

19. Alvarez-Núñez, F.A.; Yalkowsky, S.H. Relationshipbetween polysorbate 80 solubilization descriptors andoctanol-water partition coefficient of drugs. Int. J. Pharm.2000, 200, 217–222.

20. Klotarchyk, M.; Chen, S.H. Analysis of small-angle neutron-scattering spectra from polydisperse interacting colloids. J.Chem. Phys. 1983, 79, 2461–2469.

21. Itri, R.; Amaral, L.Q. Micellar-shape anisometry near isotro-pic liquid-crystal phase-transitions. Phys. Rev. E, 1993, 47,2551–2557; Erratum in E58, 1173 (1998).

22. Marignan, J.; Basserau, P.; Delord, P.J. Effect of pentanoland concentration on the micelles in the system OBS/water/n-pentanol. J. Phys. Chem. 1986, 90, 645–652.

23. Hayter, J.B.; Penfold, J. Self-consistent structural and dynamicstudy of concentrated micelle solutions. Mol. Phys. 1981, 42,109–118.

24. Hansen, J.P.; Hayter, J.B. A rescaled MSA structure factorfor dilute charged colloidal dispersions. Mol. Phys. 1982, 46,651–656.

25. Preté, P.S.C.; Gomes, K.; Malheiros, S.V.P.; Meireles, N.C.;de Paula, E. Solubilization of human erythrocyte membranesby non-ionic surfactants of the polyoxyethylene alkyl ethersseries. Biophys. Chem. 2002, 97, 45–54.

26. Atwood, D.; Florence, A.T. Physicochemical Principles ofPharmacy, 3rd ed.; The MacMillan Press: London, 2003.

27. Kawakami, K.; Oda, N.; Miyoshi, K.; Funaki, T.; Ida, Y.Solubilization behavior of a poorly soluble drug undercombined use of surfactants and cosolvents. Eur. J.Pharm. Sci. 2006, 28, 7–14.

28. Krishna, A.K.; Flanagan, D.R. Micellar solubilization of anew antimalarial drug, b-arteether. J. Pharm. Sci. 1989, 78,574–576.

29. Mukerjee, P.; Cardinal, J.R. Benzene derivatives and naph-thalene solubilized in micelles. Polarity of microenvironment,


location and distribution in micelles, and correlation withsurface activity in hydrocarbon-water systems. J. PhysChem. 1978, 82, 1620–1627.

30. Magid, L.J.; Triolo, R.; Johnson Jr, J.S. Small angle neutronscattering study of critical phenomena in aqueous solutionsof C12EO8, a nonionic amphiphile. J. Phys. Chem. 1984, 88,5730–5734.

31. Nilsson, P.; Wennerstrom, H.; Lindman, B. Structure ofmicellar solutions of nonionic surfactants. Nuclear magneticresonance self-diffusion and proton relaxation studies ofpoly(ethylene oxide) alkyl ethers. J. Phys. Chem. 1983, 87,1377–1385.

32. Caponetti, E.; Chillura-Martino, D.; Pedone, L. Partitioningof macrocycle compounds in a cationic and an anionic micel-lar solution: A small angle neutron scattering study. Lang-muir 2004, 20, 3854–3862.

33. Berr, S.S. Solvent isotope effects on alkyltrimethylammo-nium bromide micelles as a function of alkyl chain length. J.Phys. Chem. 1987, 91, 4760–4765.

34. Lusvardi, K.M.; Full, A.P.; Kaler, E.W. Mixed micelles ofdodecyltrimethylammonium bromide and didodecyldimeth-ylammonium bromide. Langmuir 1995, 11, 487–492.

35. Van Voorhis, WC. Therapy and prophylaxis of systemic pro-tozoan infections. Drugs 1990, 40, 176–202.

36. Schreier, S.; Malheiros, S.V.P.; Paula, E. Surface activedrugs: self association and interaction with membranes andsurfactants. Physicochemical and biological aspects. Bio-chim. Biophys. Acta 2000, 1508, 210–234.

37. Jumaa, M.; Muller, B.W. Lipid emulsions as a novel systemto reduce the hemolytic activity of lytic agents: Mechanismof the protective effect. Eur. J. Pharm. Sci. 2000, 9, 285–290.

38. Lichtenberg, D. Characterization of the solubilization oflipid bilayers by surfactants. Biochim. Biophys. Acta 1985,821, 470–478.

39. Strickley, R.G. Solubilizing excipients in oral and injectableformulations. Pharm. Res. 2004, 21, 201–230.

40. Guyton, A.C. Fisiologia Humana, 5th ed.; Rio de Janeiro:Interamericana, 1981.

LPDT Novel Potential Drug Against T. cruzi and Its Interaction with Surfactant Micelles

Documents

Transcript of LPDT Novel Potential Drug Against T. cruzi and Its Interaction with Surfactant Micelles