Transport properties in aqueous ethambutol dihydrochloride

International Journal of Pharmaceutics 479 (2015) 306–311

Pharmaceutical nanotechnology

Transport properties in aqueous ethambutol dihydrochloride

Luís M.P. Veríssimo a, Ana M.T.D.P.V. Cabral b, Francisco J.B. Veiga b, Sara F.G. Almeida b,M. Luisa Ramos a, Hugh D. Burrows a, M.A. Esteso c, Ana C.F. Ribeiro a,*aDepartment of Chemistry, Coimbra Chemistry Centre, University of Coimbra, 3004-535 Coimbra, Portugalb Faculty of Pharmacy, University of Coimbra, 3000-295 Coimbra, PortugalcU.D Química Física, Facultad de Farmacia, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain

A R T I C L E I N F O

Article history:Received 13 October 2014Received in revised form 21 December 2014Accepted 22 December 2014Available online 26 December 2014

Keywords:TuberculosisEthambutol dihydrochlorideDiffusion coefficientsSolutionsTransport properties1H NMR spectroscopy

A B S T R A C T

Mutual diffusion coefficients, densities and viscosities are reported for aqueous solutions of ethambutolas its dihydrochloride (EMBDHC) at finite concentrations and at 298.15 K. From these experimentalresults and by using the appropriate models (Stokes–Einstein and Hartley), the hydrodynamic radii Rh,the diffusion coefficient at infinitesimal concentration D0 and the thermodynamic factors, FT, have beenestimated, permitting us to have a better understanding of the transport behavior of ethambutoldihydrochloride in solution. Elucidation of lack of any possible drug–drug interactions in these systemswas obtained by complementary 1H nuclear magnetic resonance (NMR) spectroscopy data.

ã 2015 Elsevier B.V. All rights reserved.

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International Journal of Pharmaceutics

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1. Introduction

Ethambutol as its dihydrochloride (EMBDHC) is a first-lineantituberculosis drug with a high specificity toward Mycobacteri-um tuberculosis, and is administered in an association protocol withisoniazid, rifampicin and pyrazinamide, as currently recom-mended by international guidelines (Tomioka, 2006; Scior andGarces-Eisele, 2006; Zhao et al., 2007; Banerjee et al., 2008;Ranguelova et al., 2008; Crofts et al., 2008; Golub et al., 2008).However, the global health problems of tuberculosis (TB) on thisinfectious deadly disease and the high rate of co-infection withpersons suffering from the human immunodeficiency virus (HIV)(e.g., Golub et al., 2008) have greatly contributed to the need ofdeveloping new, affordable, anti-tuberculous drugs without cross-resistance with known antimycobacterial agents. In fact, tubercu-losis was declared a global emergency by WHO in 1993. We live,therefore, in the confluence of a double challenge, expeditious andpragmatic treatment of infected populations, and control ofresistance to treatment, bound to break the cycle of infection.However, knowledge of the mechanisms involved is still limited,

* Corresponding author. Tel.: +351 239 854460; fax: +351 239 827703.E-mail addresses: [email protected] (L.M.P. Veríssimo), [email protected]

(A.M.T.D.P.V. Cabral), [email protected] (F.J.B. Veiga), [email protected](S.F.G. Almeida), [email protected] (M. L. Ramos), [email protected] (H.D. Burrows), [email protected] (M.A. Esteso), [email protected] (A.C.F. Ribeiro).

http://dx.doi.org/10.1016/j.ijpharm.2014.12.0530378-5173/ã 2015 Elsevier B.V. All rights reserved.

both in terms of bacillary defenses and the action of drugs, and theproperties and behavior of such chemical systems are poorlyknown, even though this is a prerequisite to obtain an adequateunderstanding and solve these problems of health. In fact, fewresearchers have taken into account the transport behavior ofthese anti-tuberculous drugs in aqueous solutions (e.g., Ribeiroet al., 2009, 2010), although this is an important property for theirin vivo behavior. Concerning EMBDHC, no data on mutual diffusioncoefficients are available, namely, at 298.15 K – relevant data for invivo pharmaceutical application – as far as careful literaturesearches have shown. This paper reports experimental data formutual diffusion (interdiffusion) coefficients D, measured by theTaylor dispersion method. These studies were complemented bysome density and viscosity measurements for aqueous solutions ofEBMDHC at concentrations from (0.00 to 0.10) g dm�3 at 298.15 K,in addition to NMR spectral studies on solutions.

From the experimental data it is possible to estimate importantparameters, such as the hydrodynamic radius, Rh, apparent molarvolumes, fV, and diffusion coefficient at infinitesimal concentra-tion, D0.

In addition, the Hartley equation (Erdey-Grúz, 1974; Tyrrell andHarris, 1984) and the measured diffusion coefficients are used toestimate activity coefficients for aqueous EMBDHC, contributing toa better understanding of their thermodynamic behavior inaqueous solution at different concentrations.

We intend to both contribute to the body of knowledge of thiscritical disease, and concurrently defend the important role that

Scheme 1. Ethambutol dihydrochloride (C10H24N2O2�2HCl).

Table 2Binary mutual diffusion coefficients of (EMBDHC) in aqueous solutions at differentconcentrations, c, and at 298.15 K.

L.M.P. Veríssimo et al. / International Journal of Pharmaceutics 479 (2015) 306–311 307

studies of transport properties may have on current and futuredrug development pipelines.

2. Experimental

2.1. Reagents and solutions

Ethambutol dihydrochloride (EMBDHC) (Sigma–Aldrich, pure>99.9%, SLBF2556V, M = 277.23 g mol�1) (Table 1 and Scheme 1)was used as received without further purification. The solutions forthe diffusion measurements were prepared in calibrated volumet-ric flasks using Millipore-Q water (18.2 MV cm). The solutionswere freshly prepared and de-aerated for about 30 min before eachset of runs. The uncertainty concerning their compositions wasusually within �0.1%. For the density and viscosity measurements,solutions were prepared by direct weighing both the solute anddistilled water in a Mettler AE 240 balance with a precision of�0.0001 g (the uncertainty concerning composition was less than�0.07%).

2.2. Density measurements

The densities of EMBDHC aqueous solutions at 298.15 K weredetermined with an Anton Paar DMA5000M densimeter (precisionof 1 �10�6 g cm�3 and accuracy of 5 �10�6 g cm�3 in the ranges of0–90 �C of temperature and 0–10 bar of pressure). The uncertaintyof the results obtained is estimated to be less than 0.001%.

2.3. Viscosity measurements

Viscosity measurements of these solutions were performedwith an Ostwald type viscometer, calibrated from water, immersedinto a water-thermostat bath which temperature was controlledwithin �0.02 K by using a digital thermometer. The arithmeticmean value of four flow times for each solution was taken tocalculate such viscosity values. The measurement of the efflux timewas carried out with a stopwatch with a resolution of 0.2 s. Theuncertainty of these values was less than �0.1%.

2.4. Diffusion measurements

The Taylor dispersion method for measuring diffusion coef-ficients is well described in the literature (e.g., Barthel et al., 1996;Callendar and Leaist, 2006; Ribeiro et al., 2005, 2006; Tyrrell andHarris, 1984), and consequently we only indicate some of its mostrelevant points on the experimental determination of binarydiffusion coefficients.

This technique is based on the dispersion of a very smallamount of solution injected into a laminar carrier stream of solventor solution of different composition flowing through a longcapillary tube of length and radius 3.2799 (�0.0001) � 103 cm and0.05570 (�0.00003) cm, respectively, at T = 298.15 K (�0.01 K) in acarefully home-made air thermostat.

At the start of each run, a 6-port Teflon injection valve(Rheodyne, model 5020) was used to introduce 0.063 cm3 ofsolution into the laminar carrier stream of slightly differentcomposition. A flow rate of 0.23 cm3min�1 (corresponding to3.5 rpm of the peristaltic pump head) has been used, and was

Table 1Sample description.

Chemical name Source Purity

Ethambutoldihydrochloride(C10H24N2O2�2HCl)

Sigma–Aldrich

Mass fraction > 0.99(SLBF2556V,M = 277.23 g mol�1)

controlled by a metering pump (Gilson model Miniplus 3) to giveretention times of about 8 � 103 s. The dispersion tube and theinjection valve were kept at 298.15 K (�0.01 K) in an air thermostat.

Dispersion of the injected samples was monitored using adifferential refractometer (Waters model 2410) at the outlet of thedispersion tube. Detector voltages, V(t), were measured ataccurately timed 5 s intervals with a digital voltmeter (Agilent34401A) with an IEEE interface. Binary diffusion coefficients wereevaluated by fitting the dispersion equation

VðtÞ ¼ V0 þ V1t þ VmaxtRt

� �1=2

exp �12Dðt � tRÞ2

r2t

" #(1)

to the detector voltages. The additional fitting parameters were themean sample retention time tR, peak height Vmax, baseline voltageV0, and baseline slope V1.

The concentrations of the injected solutions (c þ Dc) and thecarrier solutions (c) differed by �0.150 mol dm�3 or less. Solutionsof different composition were injected into each carrier solution toconfirm that the measured diffusion coefficients were indepen-dent of the initial concentration difference and thereforerepresented the differential value of D at the carrier-streamcomposition.

2.5. 1NMR experiments

A solution of EMBDHC 0.20 mol dm�3, pH* 4.0, was prepared inD2O (99.9%, Aldrich) and was used as a stock solution. Theadditional solutions of concentrations 0.10, 0.050, 0.025, 0.010 and0.005 0.20 mol dm�3 were prepared by dilution of this. The pH*values quoted are the direct pH-meter readings (room tempera-ture) after standardization with aqueous (H2O) buffers. The 1Hspectra were obtained on a Bruker Avance III 400 at an operatingfrequency of 400 MHz. The methyl signal of tert-butyl alcohol wasused as external reference for 1H (d 1.3).

3. Results and discussion

3.1. Concentration dependence of density and molar volume at finiteconcentrations

The experimental density and viscosity values of EMBDHCaqueous solutions at 298.15 K are indicated in Table 2. The data of

c/(mol dm�3) D � s/(109m2 s�1)

0.0010 1.055 � 0.0030.0040 0.956 � 0.0030.0100 0.907 � 0.0020.0200 0.844 � 0.0030.0500 0.772 � 0.0010.1001 0.738 � 0.002

Table 4Thermodynamic factors, FT, for EMBDHC at T = 298.15 K.

c/(mol dm�3) FT/(10�9m2 s�1)a F’T/(10�9m2 s�1)b

0.0010 0.944 0.9530.0040 0.856 0.8650.0100 0.812 0.8240.0200 0.756 0.7710.0500 0.691 0.7150.1001 0.661 0.701

a FT = Dexp/FM.b F’T = Dexphr/FM, being hr the relative viscosity of this work (see Table 3).

308 L.M.P. Veríssimo et al. / International Journal of Pharmaceutics 479 (2015) 306–311

density, r, were linearly fitted by using a least-squares regressionmethod to obtain their dependence with concentration (Eq. (2)).That is,

r ¼ 0:9971 þ 5:841 � 10�5mR2 ¼ 0:9999 (2)

where m represents the molality.The results show very good internal consistency and the density

at infinitesimal concentration r0 shows excellent agreement withthe value found in the literature (Lide, 2007).

Apparent molar volumes, fV, calculated from Eq. (3) forEMBDHC aqueous solutions are also collected in Table 2 andshown in Fig. 2.

fV ¼ V � VH2O

m¼ Mrþ 1000

m1r� 1rH2O

!(3)

M (277.23 g mol�1) is the molar mass of the solute, V is the volumeof a solution of molality m and VH2

O and rH2O are the volume and

density of pure water, respectively.By using the Eq. (4) (Masson, 1929) and our estimated apparent

molar volumes values,

fV ¼ f0V þ S0V

ffiffiffic

p(4)

the value of the apparent molar volume at infinitesimal

concentration, f0V , and the experimental slope, S0V , were deter-

mined (f0V ¼ 168:88ð�2Þcm3mol�1 and S0V ¼ 526:35cm9=2mol�3=2,

respectively).

From the positive value found for S0V, we can consider that forthe dilute aqueous system EMBDHC (c � 0.01 mol kg�1), thehydrophilic interactions (solute–water interactions) are mostsignificant when compared to the hydrophobic interactions(solute–solute interactions).

3.2. Concentration dependence of viscosity at finite concentrations

Table 3 shows the results of the viscosity of EMBDHC from 0.00to 0.099 mol kg�1. The following polynomial in c1/2 was used to fitour data by a least squares procedure

h ¼ 0:8977 þ 0:0088m1=2 þ 0:4382mR2 ¼ 0:9997 (5)

where m and h represent the molality concentration and theviscosity of EMBDHC in different aqueous solutions, respectively.The goodness of the fit (obtained with a confidence interval of 98%)was assessed by the excellent correlation coefficient, R2, and thelow standard deviation (<1%). Moreover, the deviation betweenthe limiting h0 value calculated by extrapolating experimentalresults to c ! 0 and the measured value for solvent is alsoacceptable (<1.0%).

The analysis of the dependence of the viscosity on theconcentration was evaluated by fitting the values of relativeviscosity, hr, (Table 4) to the Jones–Dole equation (Jones and Dole,1929) as follows,

Table 3Experimental density, s, viscosity data, h, and apparent molar volumes of EMBDHCin aqueous solutions at different concentrations, m, and at 298.15 K.

103m/(mol kg�1) r/(g cm�3) 106sa fV/(cm�3mol�1) h/mPa s 106s

0.000000 0.997048 0.8902 0.160.525648 0.997141 0.00 100.07 0.8977 0.181.068824 0.997145 4.78 186.74 0.8983 0.022.065847 0.997225 0.79 191.83 0.8987 0.035.044478 0.997406 2.26 206.59 0.9011 0.53

10.01862 0.997710 3.95 211.45 0.9029 0.0349.89092 0.999995 2.90 217.99 0.9213 0.0499.86323 1.002932 0.54 217.50 0.9443 0.23

a s representa o desvio padrão para as medidas.

hh0

¼ 1 þ Ac1=2 þ Bc þ Dc2 (6)

where c is the concentration (g dm�3), and A and B are empiricalterms. The A coefficient has been related to the long distancesolute–solute interactions taking place in the solution and the Bcoefficient has been found to depend upon solute–solventinteractions. The coefficient D addresses both solute–solute andsolute–solvent interactions and it becomes important only at highsolute concentration and was, therefore, disregarded for thisanalysis in Eq. (6) (Donald et al., 1995).From the A value close tozero (A = 0.01 dm3/2mol�1/2) and the low positive B value (B = 0.49dm3mol�1) (Table 4), we can strongly suggest that the interactionsbetween EMBDHC and water are the predominant ones, with thisdrug acting as a structure-making species. In contrast, drug–druginteractions are suggested to be relatively weak. As we will seeshortly, this is supported by NMR studies on the system.

3.3. Concentration dependence of diffusion coefficient at finiteconcentrations

Table 2 gives the average experimental values for the mutualdiffusion coefficient, D, with value for each carrier solutiondetermined from 4 to 5 profiles generated by injecting samplesthat were more or less concentrated than the carrier solution. Goodreproducibility was, in general, observed, with results within �2%.

The concentration dependence of the measured diffusioncoefficients can be represented by the polynomial equation:

D ¼ 1:117 � 2:530c1=2 þ 4:248cðR2 ¼ 0:992Þ (7)

The goodness of the fit (obtained with a confidence interval of98%) can be assessed by the excellent correlation coefficients, R2,and the low percentage of standard deviation (1%).

The interpretation of the diffusion behavior of these aqueoussystems on the basis of the Nernst–Hartley equation (Tyrrell andHarris, 1984), suggests that two different effects, can control thediffusion process: the ionic mobility at infinitesimal concentrationand the gradient of the free energy. On the other words, D is aproduct of both kinetic, FM (or molar mobility coefficient of adiffusing substance) and a thermodynamic factor,

D ¼ FMFT (8)

where

FM ¼ D0 (9)

FT ¼ 1 þ c@lny�@c

� �(10)

D is the mutual diffusion coefficient of the electrolyte in m2s�1, D0

is the mutual diffusion coefficient of the solute at infinitesimalconcentration, and the last term in parenthesis is the activity

0.90

0.95

1.00

1.05

0.0 0 0.0 2 0.0 4 0.06 0.08 0.1 0

(Dη

/T) re

l

c/mol k -1

Fig.1. (Dh/T)rel values of for EMBDHC aqueous solutions at 298.15 K, as a function ofthe medium concentration, c.

L.M.P. Veríssimo et al. / International Journal of Pharmaceutics 479 (2015) 306–311 309

factor, with y� being the mean molar activity coefficient and c theconcentration in mol m�3.

In this equation, phenomena such as ion association (Tyrrelland Harris, 1984; Robinson and Stokes, 1959) and viscosity are nottaken into consideration. Thus, considering Eqs. (8)–(10) and ourdata, we have estimated the thermodynamic factor values for theinterval of concentrations where, in general, these equations arevalid (i.e., c � 0.01 mol kg�1, Table 3). The analysis of the datashown in the second column of Table 3 indicates that the gradientof the free energy (FT) decreases with the increase in concentration.The decreasing of the gradient of the free energy with concentra-tion, FT, leads us to assume the presence of solute–soluteinteractions as being responsible by these facts. Thus, consideringour experimental conditions (i.e., dilute solutions), and conse-quently, assuming that there are no changes in the effectivemobilities of the ion species (Cl� and EMBH2)2+) caused by theelectrophoretic effect, and that some parameters, such as viscosity,dielectric constant, hydration and association, do not change withconcentration, we can conclude that the variation in D is mainlydue to the variation of FT (attributed to non-ideality in thethermodynamic behavior).

Considering now the influence of the viscosity factor (Tyrrelland Harris, 1984) on the behavior diffusion of EMBDHC in aqueoussolutions at finite concentrations, we have estimated a new F’Tvalues (the third column of Table 3) as equal to

F0T ¼ FThh0

� �(11)

where h0 and h represent the viscosity of water and the solution,respectively. These F’T values are, obviously, higher than those of FTcalculated from Eq. (10), with the discrepancies becoming moresignificant when the concentration increases. This effect ofincreasing of the thermodynamic factor makes the contributionof the FM factor to be even smaller when the concentration ofEMBDHC becomes higher.

3.4. Tracer diffusion coefficient of the (EMBH2)2+ ion and mutualdiffusion coefficient of EMBDHC at infinitesimal concentration

Table 2 gives the experimental values for the mutual diffusioncoefficient when the carrier stream and the injection samples aresolutions at different concentrations. The value at infinitesimalionic strength, D0, estimated by extrapolating to c ! 0 isD0 = 1.117 x 10-9m2 s-1.

By using the Nernst–Hartley equation (Robinson and Stokes,1959),

D0 ¼ RT

F2jZðEMBH2Þ2þj þ jZCl� jl0

ðEMBH2Þ2þl0Cl�

jZðEMBH2Þ2þZCl� jl0ðEMBH2Þ2þ þ l0

Cl�(12)

and assuming that the above D0 coincides with the Nernst valueand that the diffusion of this drug is treated as the diffusion of thepredominant species (EMBH2)2+ and Cl� ions (fact supported by

Table 5Hydrodynamic radius, Rh, of EMBDHC solutions of concentrations, c, and differentviscosities, h, at 298.15 K.

103m/(mol kg�1) Rh/(nm) (Dh/T)rel

0.000000 2.041 1.00000.52565 2.025 1.00801.06882 2.026 1.00742.06585 2.028 1.00625.04448 2.032 1.0041

10.0186 2.044 0.998349.8909 2.126 0.960099.8632 2.211 0.9232

pKa1 = 6.35 and pKa2 = 9.35 of protonated ethambutol values (Beggs

and Andrews, 1974), we estimated l0ðEMBH2Þ2þ = 44.15 �10�4m2V�1

mol�1 for 298.15 K. ZCl� and Z(EMBH2)2+ represent the algebraic

valences of Cl� anion and (EMBH2)2+ of a cation, respectively. l0Cl�

is the equivalent conductance of Cl� at infinitesimal concentration,given in the literature at 298.15 K (Robinson and Stokes) as

l0Cl� = 76.35 �10�4m2V�1mol�1 (Robinson and Stokes, 1959).

From the l0ðEMBH2Þ2þ value, and assuming that the limiting tracer

diffusion coefficient of the (EMBH2)2+ ion, D0T , can be estimated

through the Nernst equation (Eq. (13)),

l0ðEMBH2Þ2þ ¼ D0

TF2ZðEMBH2Þ2þ

RT(13)

the limiting tracer diffusion coefficient value, D0T = 0.588 � 10�9

m2 s�1, was obtained for the (EMBH2)2+ ion.Our calculation shows that the mutual diffusion coefficient of

the EMBDHC at infinitesimal ionic strength is significantly larger(1.9 times) than that of the corresponding tracer diffusioncoefficient for the (EMBH2)2+ ion, which suggests the electrostaticdragging effect of the chloride ions on the (EMBH2)2+ ion diffusion.The effect of the interactions between (EMBH2)2+ and Cl� ions, canbe responsible for the decrease in the frictional resistance of thatdrug that acts toward the increase of its mutual diffusioncoefficient. However, the relatively low diffusion of the(EMBH2)2+ ion indicates that this has a strong structure-makingeffect on water.

Thus, from the analysis of the dependence of our data ofviscosity, apparent molar volume, and diffusion coefficient on theconcentration, although we can consider that all types ofinteraction may occur between the drug and water, it is probablethat the solute–water interactions are the predominant ones.

3.5. Hydrodynamic radius of EMBDHC

From the Stokes–Einstein equation (Eq. (14)) (Erdey-Gruz,1974), which considers the solvent as a continuum characterized

CH3CH2CHN

H

CH2CH3 CH2NCHCH2OHH

CH2OH

CH2

(2)(1) (1'')

(2') (1')(3')

(4')

(4'')

(2'') (3'')

Scheme 2. Structure and the numbering of EMBDHC used for labeling the spectra.

Fig. 2. 1H NMR spectra of D2O solutions of EMBDHC (i) 0.20, (ii) 0.10, (iii) 0.050, (iv) 0.025, (v) 0.010 and (vi) 0.005 mol dm�3, pH* 4.0, temperature 298.15 K.

310 L.M.P. Veríssimo et al. / International Journal of Pharmaceutics 479 (2015) 306–311

by its bulk viscosity value, it is also possible to estimate thehydrodynamic radius, Rh, of EMBDHC,

D0T ¼ KBT

6ph0Rh(14)

where D0T , Rh, KB and h0 are the self-diffusion coefficient at

infinitesimal concentration, the hydrodynamic radius of anequivalent spherical particle, the Boltzmann’s constant and theviscosity of the solvent at temperature T, respectively.

By taking into account the Stokes equation (Eq. (13)) andreplacing the water viscosity by the viscosity of the solutions, thevalues of the effective hydrodynamic radius Rh of EMBDHC havebeen estimated as a function of the concentration of the solute andcollected in Table 5. The maximum variation observed in these Rhvalues for m < 0.01 mol kg�1, with respect to the limiting one atinfinitesimal concentration, is around 1%, which is close to theimprecision of the diffusion measurements (<3%).

Eq. (14) can be rearranged in the form

Dh

T

� �rel ¼ ðDh=TÞ

ðD0h0=TÞ(15)

showing a reciprocal dependence between the grouping (Dh/T)and the effective hydrodynamic radius Rh. If this radius keepsconstant when the medium viscosity changes, the right-hand sidein Eq. (14) ought to be constant, which would mean that thediffusion process is solely controlled by the viscosity of themedium. Table 5 and Fig. 1 give the (Dh/T)rel values. As can be seen,such constancy is not observed but, in contrast, a constantdecrease of these values with concentration is observed. Thisbehavior indicates that the decrease in D with concentration is notadequately compensated by the viscosity increase of the medium,and, hence, it would be necessary to take into account any othereffects. This behavior can be attributed to the limitations of theStokes–Einstein relation, where some phenomena are notconsidered (e.g., the shape of the solute molecule is far frombeing spherical and the replaced viscosity value concerns the bulksolution instead of the local one in the neighborhood of the solutemolecules, whose presence may affect the solvent structure and,consequently, its viscosity).

3.6. 1NMR studies as a function of the concentration of EMBDHC

Further information on solute interactions can be obtained byNMR spectroscopy. Scheme 2 shows the structure and thenumbering of EMBDHC used for labeling the spectra, and Fig. 2shows the 1H NMR spectra, for solutions 0.20, 0.10, 0.050, 0.025,0.010 and 0.005 mol dm�3, respectively, prepared by dilution fromthe most concentrated one (pH 4.0).

Both the 1H chemical shift and the linewidth of each signal werefound not to change with increasing concentration, suggesting nodrug–drug aggregates or interactions under these conditions, incomplete agreement with the transport property studies. Thisresult is not unexpected, as, according to the pKa values of the ofN–H2

+ groups of the drug, (NH2+! N–H) (pK1 = 6,35, pK2 = 9.35),

EMBDHC is fully protonated at pH 4.

4. Conclusions

We have measured mutual diffusion coefficients, densities andviscosities of binary aqueous solutions of EMBDHC at 298.15 K.Considering the results obtained for the diffusion coefficient,possible solute–solute electrostatic interactions can be present,resulting in a decrease in the diffusion coefficients and thermody-namic factors. However, the relatively low diffusion of the(EMBH2)2+ ion indicates that this ion has a strong structure-making effect on water, and that such interactions are negligible.This is supported by NMR studies, which suggest no drug–druginteractions up to 0.20 mol dm�3 EMBDHC, and the analysis of thevariation of both the apparent molar volume and the viscosity ofthese solutions with the concentration. In fact, from the positive

value found to S0V , from the A value close to zero (A = 0.01 dm3/

2mol�1/2) and the low positive B value (B = 0.49 dm3mol�1), it canbe concluded that solute–solvent interactions are the predominantones, in complete agreement with the NMR results.

In conclusion, from the analysis of all data, although all types ofinteraction may occur with EMBDHC in water, the results stronglysuggest that solute–water interactions are the predominant ones,and that the drug exists as isolated molecules in solution underthese conditions. We thus believe that transport data measured foraqueous solutions of EMBDHC in aqueous media provide

L.M.P. Veríssimo et al. / International Journal of Pharmaceutics 479 (2015) 306–311 311

information necessary to model diffusion in pharmaceuticalapplications.

Acknowledgements

Financial support of the Coimbra Chemistry Centre from the FCTthrough project Pest-OE/QUI/UI0313/2014 is gratefully acknowl-edged.

NMR data was obtained at the UC-NMR facility which issupported in part by FEDER – European Regional DevelopmentFund through the COMPETE Programme (Operational Programmefor Competitiveness) and by National Funds through FCT –

Fundação para a Ciência e a Tecnologia (Portuguese Foundationfor Science and Technology) through grants REEQ/481/QUI/2006,RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER-002012, andRede Nacional de Ressonância Magnética Nuclear (RNRMN).

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