International Journal of Pharmaceutics 474 (2014) 8090

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Effect of g-cyclodextrin on solubilization and complexation of

irbesartan: Inuence of pH and excipients
Chutimon Muankaew a,b , Phatsawee Jansook c, Einar Stefnsson d, Thorsteinn Loftsson a, *
a

Faculty of Pharmaceutical Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavik,Iceland

Faculty of Pharmacy, Siam University, 38 Petkasem Road, Phasicharoen, Bangkae, Bangkok 10160, Thailand
c
Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand
d
Department of Ophthalmology, Faculty of Medicine, National University Hospital, Eirksgata 37, IS-101 Reykjavk, Iceland
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 25 June 2014
Received in revised form 7 August 2014
Accepted 9 August 2014
Available online 13 August 2014

In effort to prepare an eye drop formulation of irbesartan, the effect of g-cyclodextrin complexation on
irbesartan solubilization in aqueous solutions was investigated. The optimum cyclodextrin concentration
for formation of irbesartan/cyclodextrin inclusion complex was found to be 10% (w/v) and the solubility
of ionized irbesartan/g-cyclodextrin complex (at pH 7.2) was shown to be three fold greater than that of
the unionized complex (at pH 4.3). The irbesartan ux through semipermeable membranes increased
with increasing g-cyclodextrin concentration at both pH values. However, the ionized complex displayed
decrease in the drug permeation coefcient with increasing cyclodextrin concentration. The effect of four
pharmaceutical excipients on the cyclodextrin solubilization was investigated. EDTA, hydroxypropyl
methylcellulose, and tyloxapol increased complexation efciency of g-cyclodextrin while benzalkonium
chloride had negligible effect. The largest solubilization was observed in the eye drop vehicle that
contained all four excipients in addition to g-cyclodextrin. Dynamic light scattering measurements
disclosed that excipients had impact on size of complex aggregates and consequently on the drug ux
through the semipermeable membranes. Complex of irbesartan/g-cyclodextrin was characterized by
FT-IR, 1H NMR, XRPD, and TEM techniques.
2014 Elsevier B.V. All rights reserved.

Keywords:
Irbesartan
g-Cyclodextrin
Solubilization
Complexation
pH
Excipient

1. Introduction
The renin-angiotensin system in ocular tissue has been
extensively studied in recent years and detection of angiotensin
II receptor type 1 in human eyes indicated that this local receptor
may be involved in the regulation of intraocular pressure (IOP)
(Meka et al., 2012). Some publications have reported that
angiotensin II receptor blockers (ARBs) possess IOP lowering
effect (Clermont et al., 2006; Fujita et al., 2012; Yoshida et al., 2004;
Zhang et al., 2012). Irbesartan, a selective AT1 subtype angiotensin
II receptor antagonist, was selected as a model drug in this study.
The drug is a biopharmaceutical class II drug possessing low
aqueous solubility and high membrane permeability. The drug is
mainly given orally in the treatment of cardiovascular diseases.
Due to its limited solubility, several formulation techniques have
been tested in an effort to enhance irbesartan dissolution rate, its
absorption and bioavailability after oral administration such as

* Corresponding author. Tel.: +354 525 4464; fax: +354 525 4071.
E-mail address: thorstlo@hi.is (T. Loftsson).
http://dx.doi.org/10.1016/j.ijpharm.2014.08.013
0378-5173/ 2014 Elsevier B.V. All rights reserved.

formation of solid dispersions (Aruna et al., 2011), formation of

irbesartan/b-cyclodextrin complexes (Hirlekar and Kadam, 2009;
Hirlekar et al., 2009), particle size reduction (Jain and Kurup, 2013;
Zhang et al., 2011) and self-emulsifying technique (Patel et al.,
2011). To the best of our knowledge, few studies have been
performed in order to improve topical delivery of irbesartan from
aqueous eye drop solutions or suspensions (Jansook et al., 2014). It
is well known that conventional eye drop formulations are not
effective drug delivery systems. The main reasons are the
complicated anatomy structure of the eye and rapid drug clearance
from the eye surface resulting in less than 5% topical bioavailability
(Kavitha et al., 2013; Madhuri Divya et al., 2013). Formulation of
ophthalmic drugs as aqueous drug/cyclodextrin nano- and micro
suspensions has resulted in dramatic improvement in their topical
bioavailability (Johannesson et al., 2013; Loftsson et al., 2007b).
Here the effects of g-cyclodextrin on the irbesartan solubility,
nanoparticle formation and irbesartan delivery through articial
membranes were investigated.
Cyclodextrins (CDs) are truncate cone oligosaccharides with
hydrophilic external surface and a somewhat hydrophobic
internal cavity. CDs are commonly used pharmaceutical

C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

excipients and can be found in close to 40 different pharmaceutical products (Kurkov and Loftsson, 2013). CDs can act as
complexing agents that increase aqueous solubility of hydrophobic drugs and as penetration enhancer that promote drug
permeation and absorption across membrane barrier thus
improving drug bioavailability (Acarturk and Celebi, 2011; Carrier
et al., 2007; Loftsson and Duchene, 2007). In many cases, CDs are
utilized to prevent interactions between active pharmaceutical
ingredients (API) and excipients, to enhance drug stability and to
reduce drug irritation. Furthermore, CDs have been employed as
carrier systems to deliver APIs to their targets. gCD and some
water-soluble CD derivatives, such as 2-hydroxypropyl bCD and
sulfobutyl ether bCD, have been applied in eye drop formulations
(Abd El-Bary et al., 2011; Granero and Longhi, 2010; Jansook et al.,
2010b; Loftsson et al., 2007b). For example, gCD can be used to
improve aqueous solubility and bioavailability of drug molecules
through inclusion complex formation. gCD shows less phospholipid membrane extraction in comparison to other natural CDs
and toxicity studies have revealed that it is well tolerated both
after oral and parenteral administration (Monnaert et al., 2004;
Munro et al., 2004) as well as after topical administration to the
eye (Gudmundsdottir et al., 2014; Johannesson et al., 2014;
Johannesson et al., 2013; Tanito et al., 2011). Microparticulate
formulations such as liposomes and CDs have been shown to
enhance penetration of lipophilic drugs into and through skin
(Gillet et al., 2009; Gillet et al., 2011; Konradsdottir et al., 2009).
Recent publications show that CD based eye drop vehicles can
increase topical drug delivery to both the anterior and posterior
part of the eye. Increasing the size of the nanoparticles in
ophthalmic drug formulation can prevent drug elimination form
the eye surface and produce high sustained drug concentrations
in the tear uid (Loftsson, 2013; Loftsson et al., 2007b; Loftsson
et al., 2002).
Benzalkonium chloride is commonly used preservative in
ophthalmic preparations, it is also known to act as surface active
agent and enhance topical drug penetration. Likewise, disodium
edetate is employed to increase antimicrobial effect of benzalkonium chloride in aqueous medium (Sklubalova, 2004). Previous
studies have shown that addition of small amount water soluble
polymer such as PVP, poloxamer, and cellulose derivatives can
improve drug solubility and complexation efciency of CDs toward
poorly soluble guest molecules (Loftsson and Frioriksdottir, 1998;
Loftsson and Masson, 2004; Taupitz et al., 2013). The purpose of
this present study was to investigate the inuence of gCD and the
combined effect of pH and several pharmaceutical excipients on
solubilization and gCD complexation of irbesartan in order to be
able to select suitable conditions for an optimal irbesartan eye drop
formulation. The physicochemical properties of irbesartan and
excipients tested shown in Table 1.
2. Materials and methods
2.1. Materials
Irbesartan was purchased from Sun Mil Compound (Mumbai,
India), g-cyclodextrin (gCD) was purchase from Wacker (Surrey,
UK). Disodium edetate dihydrate (EDTA) was purchased from
Merck (Darmstadt, Germany). Hydroxypropyl methylcellulose
(HPMC) benzalkonium chloride (BAC) and tyloxapol were purchased form SigmaAldrich (St. Louis, MO, USA). Cellophane
membrane with molecular weight cut of (MWCO) 3500 Da, 6000
8000 Da, and 12,00014,000 Da (Spectra/Pore1) were purchased
from Spectrum Laboratories Inc. (Rancho Dominquez, USA). All
other reagents used were analytical grade. Milli-Q water (Millipore, Billerica, MA) was used for sample preparation and all
solution.

81

2.2. Characterization of complexes

2.2.1. Dynamic light scattering (DLS)
Particle sizes of gCD complexes in aqueous solutions were
determined by DLS using Nanotrac Wave particle size analyzer
(Microtrac Inc. USA). Wavelength of the laser was 780 nm and the
scattering angel was 180
. The intensity of scattering light
generated by Brownian movement of particles was measured,
and then the velocity and the diffusion coefcient (D*) were
determined. The hydrodynamic diameter (d) of the particles was
calculated from the apparent diffusion coefcient applying the
StokesEinstein equation;
d

kT
3p hD

(1)

where d is the hydrodynamic diameter (m), k is the Boltzmann

constant (J/K), T is the temperature in Kelvin, h is the solvent
viscosity (kg/(m s)) and D* is the diffusion coefcient (m2/s). The
size population of the aggregates was estimated by using the
following equation:
A =Ra
Mi P i i a  100
Ai =Ri

(2)

where Mi is the mass distribution percentage, Ai is the intensity

area, Ri is the hydrodynamic radius of the size population i (Bonini
et al., 2006; Gonzalez-Gaitano et al., 2003) and a is the shape
parameter that equals three, assuming spherical shaped particles.
The samples were ltered through 0.45 mm membrane lter before
particle size determination. Each measurement was done in
triplicate at controlled temperature, 25  0.2
C.
2.2.2. Fourier transform infrared (FT-IR)
FT-IR spectra of complexes were recorded on Thermo Nicolet
Avatar 370 FT-IR spectrometer (Thermo Scientic, Madison, USA)
in the range of 4004000 cm1 with resolution of 4 cm1 for each
sample. The pellets were prepare by mixing sample with KBr and
then compressed to obtain self-supporting disk. The measurements were conducted at room temperature.
2.2.3. 1H NMR spectroscopic studies
1
H NMR spectra of complexes were recorded on Brucker
AVANCE 400 instrument (Brucker Biospin GmbH, Karlsruhe,
Germany) at 500 MHz. Solutions of gCD, irbesartan/gCD complex
and irbesartan solution were prepared separately in deuterated
dimethyl sulfoxide (DMSO-d6). The magnitude of chemical shift
was expressed in ppm (d).
2.2.4. X-ray powder diffraction (XRPD) studies
X-ray powder diffractometry (XRPD) identication of the
crystal internal structure was determined by XRPD (Miniex II,
Rigaku, Japan). Wide angle XRPD using CuKa radiation at 40 kV
and 20 mA was employed. The scan speed and angular scan range
was set at 1
per min and 540
2u, respectively. 1:1 and 1:2 molar
ratios of drug and gCD were prepared by lyophilizer at 52
C for
48 h and identical physical mixture of drug and gCD were prepared
by blending in mortar.
2.2.5. Transmission electron microscopy (TEM) analysis
Particle morphology and size of aggregates in irbesartan
saturated aqueous 10% (w/v) gCD and eye drop vehicle were
analyzed using Model JEM-1400 transmission electron microscope
(JEOL, Tokyo, Japan) after negative staining of the sample by uranyl
staining method (Bgler et al., 1998). The preparation was done
under constant vacuum, briey; formvar-coated grids were oated
on a droplet of sample preparation on paralm to permit the

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C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

Table 1
The physicochemical properties of irbesartan and the excipients tested (Brewster and Loftsson, 2007; Jansook and Loftsson, 2009; Kaur et al., 2000; Muszalska et al., 2014).
Physicochemical properties

Chemical structure

Molecular
weight

Melting point
(
C)

Log Po/w

pKa

S0 (mg/ml) in water

Irbesartan

428.53

180182

10.1 (at pH
7.4)

4.12; 7.40

2.7  104

gCD

1297.1

200

12

232

Benzalkonium chloride

360

40

Very soluble

Edetate disodium

336.2

252

2.0; 2.7; 6.2;

10.3

96

Hydroxypropyl
methylcellulose

10,000
1,500,000

190200

Varies with the molecular

weight

Tyloxapol

298.4

Miscible

adsorption of the particles onto the grid. The grid was blotted with
a lter paper and transferred onto a drop of negative stain, 2%
aqueous uranyl acetate solution.
2.3. Phase-solubility studies
The solubility of irbesartan under the various conditions was
determined by a heating method as previously described (Loftsson
et al., 2005). Briey, excess amount of drug was added to aqueous
gCD solutions to form suspensions. The suspensions were heated
in autoclave in sealed vials at 121
C, for 20 min, and allowed to cool

at room temperature (2223
C). Then, a small amount of solid

drug was added to each suspension. The vials were resealed, placed
in a shaker (KS 15 A Shaker, EB Edmund Bhler GmbH, Germany)
and allowed to equilibrate at room temperature (2223
C) under
constant agitation for 7 days. Before equilibration the pH was
adjusted to either 4.3  0.05 or 7.2  0.05 (Thermo Orion 3 StarTM
benchtop pH meter, Thermo Fisher Scientic, USA) using
concentrated hydrochloric acid or sodium hydroxide solutions
and the pH measured periodically. After equilibrate was attained,
the suspensions were ltered through 0.45 mm membrane lter
(Spartan 13/Whatman, Germany). The ltrate was diluted with

C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

mobile phase and analyzed by HPLC. Each experiment was

performed in triplicate and the results reported as the mean
values  standard deviation (SD).
Phase-solubility prole was determined according to the
method of Higuchi and Connors (Higuchi and Connors, 1965).
The apparent stability constant values (K1:1) (Eq. (3)) and the
complexation efciencies (CE) (Eq. (4)) were calculated from the
slopes of linear phase-solubility diagrams.
K 1:1

slope
s0 1  slope



slope
Drug=gCD complex

CE
1  slope
gCD

3. Results and discussion

3.1. Characterization of irbesartan/g CD inclusion complexes
(4)

2.4. Quantitative determination method

The concentration of irbesartan was determined using
reversed-phase high performance liquid chromatography (HPLC)
(Dionex, Softron GmbH Ultimate 3000 series, Germany). The
system consisted of a P680 pump with a DG-1210 degasser, an ASI100 autosampler and VWD-3400 UVvis detector. Phenomenex
Luna C18(2) 150 mm  4.6 mm, 5 mm column with matching guard
column (Phenomenex, UK) and was operated under isocratic
condition and ambient temperature. The mobile phase consisted of
methanol and aqueous 0.05% (v/v) phosphoric acid solution
(70:30 v/v). The injection volume was 20 ml, the ow rate
1 ml/min and the UV detector was operated at 244 nm.
2.5. Permeation studies
The effect of gCD with or without excipients on irbesartan
permeation through semi-permeable membranes was experimented using unjacketed Franz diffusion cells (SES GmbH
Analysesysteme, Germany) with diffusion area of 1.77 cm2.
Phosphate buffer saline solution pH 7.4 containing 2% (w/v)
gCD was used as receptor phase solution. The receptor phase
(12 ml) was sonicated under vacuum to remove dissolved gas.
The sample (1.5 ml) was added to the donor chamber after
ltration through 0.45 mm membrane lter. The sample and the
receptor phase was separated by a single layer of semipermeation membrane (MWCO 3500 Da, 60008000 Da, or
12,00014,000 Da) that had been soaked overnight in the
receptor phase solution. The study was conducted at ambient
temperature under continuous stirring of the receptor phase
solution using magnetic stirring bar rotating at 300 rpm. A 150 ml
of receptor phase was collected at 30, 60, 90, 120, 180, and
300 min and immediately replaced with fresh receptor phase.
The drug concentration was analyzed by HPLC. The steady state
ux was calculated as the slope (dq/dt) of linear section of the
amount of drug in the receptor chamber (q) versus time (t)
proles, and the apparent permeability coefcient (Papp) was
calculated from the ux (J) according to Eq. (5):
dq
Papp  C d
A  dt

experiment was performed in triplicate and the results reported as

the mean values  standard deviation (SD).
The surface tension of drug in 10% (w/v) gCD aqueous solution
composed of different excipient and in mixture was measured by
plate method using Krss Tensiometer K9 (Krss GmbH, Germany).
The experiment conducted at 25  0.2
C, each samples was
determined three times.

(3)

where S0 is the intrinsic solubility of drug in the complexation

media.
In addition, solubility of irbesartan in aqueous 020% (w/v) gCD
solution pH of 7.2  0.05 containing 0.02% (w/v) benzalkonium
chloride, 0.1% (w/v) EDTA, 0.25% (w/v) HPMC and/or 0.1% (w/v)
tyloxapol was determined as describe above. Aqueous solution
containing all the excipients constitutes the aqueous eye drop
vehicle.

83

(5)

where A is the surface area of mounted chamber (1.77 cm2) and Cd

is initial concentration of irbesartan in the donor phase. Each

FT-IR spectra of irbesartan, gCD and the irbesartan/gCD

complex are displayed in Fig. 1. The spectrum of irbesartan has
characteristic peaks at 3430 cm1 (N
H stretch), 3057 cm1
(aromatic C
H stretch), 2961, 2929, 2869 cm1 (aliphatic C
H
stretch) and 741 cm1 (1,2-di-substituted benzene) that disappeared upon complexation and the frequencies of the gCD spectra
shifted to higher frequency. This observation indicates formation
of irbesartan/gCD complexes.
The interaction between irbesartan and gCD was investigated
by 1H NMR and the difference in the chemical shifts (Dd) between
free and bound gCD molecules is shown in Table 2. H-3 and
H-5 protons are located in the interior of CD cavity while others
(H-1, H-2H-4, and H-6) are located on the outer surface of CD
molecule (Lehmann et al., 1991; Rajendiran and Siva, 2014). The
H-3, H-5, H-1, and H-6 protons Dd showed slightly up eld shifts
whereas H-2 and H-4 protons showed insignicant change. This
result indicated formation of inclusion complex between irbesartan and gCD.
The powder diffraction pattern of the lyophilized irbesartan/
gCD complex, physical mixture of irbesartan and gCD, pure gCD,
and pure irbesartan are shown in Fig. 2. The combined peaks of
drug as well as gCD were presented in PXRD diffractogram of the
physical mixture with reduced intensities while characteristic
peaks of irbesartan and gCD had virtually disappeared from the
lyophilized complex diffractogram. Thus, it was assumed that drug
had converted from crystalline to another form as a result of the
inclusion complex formation (Chawla and Bansal, 2007; Hirlekar
and Kadam, 2009; Negi and Singh, 2013).
Transmission electron microscope image of irbesartan saturated aqueous 10% (w/v) gCD compared to irbesartan saturated eye
drop formulation is shown in Fig. 3. Small amounts of irbesartan/
gCD complex aggregates are observed in both samples, however,
the size of aggregates in eye drop vehicle is larger than that of
aqueous gCD solution. These are related to drug solubility and
permeability coefcient results as discussed above.
3.2. Effect of g CD and pH on phase solubility studies
Phase-solubility proles of irbesartan in pure aqueous gCD
solution at two different pH (4.3 and 7.2), and the eye drop vehicle
are presented in Figs. 4 and 5. Increasing gCD concentration
increases the irbesartan solubility up to a plateau at 10% (w/v) gCD.
Hence, the phase-solubility proles were classied as BS type
according to HiguchiConnors classication system (Higuchi and
Connors, 1965). This indicates that irbesartan forms inclusion
complex with gCD but that the complexes has limited solubility in
the complexation medium. The inuence of pH on the complex
formation was also determined. Irbesartan, a weakly acid drug, has
been reported to have pKa1 of 4.12 and pKa2 of 7.40 (Demiralay
et al., 2010; Vujic et al., 2012). At pH 4.3, irbesartan is partly
deprotonated (i.e., unionized) and partly protonated (i.e., in its
cationic form). However, at pH 7.2 irbesartan is predominantly in
its anionic form. The K1:1 was found to be 450 M1 and the CE is
0.03 at pH 4.3 while the K1:1 was found to be 230 M1 and the CE is

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C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

Fig. 1. FT-IR spectra of raw irbesartan (A), gCD (B) and irbesartan/gCD inclusion complex (C).

0.06 at pH 7.2. Although irbesartan exhibits greater afnity for gCD

in its unionized form (pH 4.3) the low CE value at that pH indicates
that a larger number of gCD molecules are required to form a
irbesartan/gCD complex at pH 4.3 than at pH 7.2 (Loftsson et al.,

2005, 2007a). The optimal amount of gCD needed to dissolve and

form complexes with irbesartan is about 10% (w/v) at pH 7.2, and
the irbesartan solubility is three fold greater at that pH than at pH
4.3.

Table 2
1
H NMR Chemical shift corresponding to gCD in free-state and in complex.
Proton

gCD

Irbesartan/gCD

Dd (ppm)a

Comment

H-1
H-2
H-3
H-4
H-5
H-6

4.8516
3.3350
3.5860
3.2769
3.5066
3.4820

4.8604
3.3093
3.5951
3.3078
3.5661
3.4911

0.0056
0.0073
0.0101
+0.0054
0.0131
0.0077

Up eld
Down eld
Up eld
Down eld
Up eld
Up eld

Dd is difference in chemical shift values calculated from d(gCD)  d(irbesartan/gCD).

C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

85

Fig. 2. XRPD spectra of lyophilized irbesartan/gCD (1:1) and/or (1:2) complex, physical mixture of irbesartan and gCD (1:1) and/or (1:2), pure gCD and pure irbesartan.

Fig. 3. Transmission electron microscopic images of irbesartan/gCD complex aggregates in 10% (w/v) cyclodextrin aqueous solution (A) and eye drop vehicle (B).

3.3. Effect of g CD and pH on permeation ux and fraction of

aggregates
The ux proles of irbesartan in aqueous gCD solutions are
shown in Fig. 6, the greater ux at high pH is a result of increased
drug solubility. However, the graphs level off after 5% (w/v) of gCD
at both pH values due to formation of water-soluble complex
aggregates. The estimated fraction of aggregate complexes in the
aqueous gCD solutions was calculated from Eq. (6) as previously
described by Messner et al. (Messner et al., 2011a):
fA  1 

Fig. 4. The phase-solubility diagram of irbesartan in 020% (w/v) gCD aqueous

solution at pH 4.3  0.05 (^) and 7.2  0.05 (&). The effect of pH on drug solubility
was statistically signicant; P < 0.05, student t-test.

Jexp
Jthe

(6)

where Jexp is the experimental ux and Jthe is the theoretical ux

obtained by extrapolation from the linear part of the graph (i.e., at
[gCD] 5%); the results are presented in Fig. 7. At pH 7.2, increasing
of the gCD concentration lead to increased aggregation and the
aggregate diameter also tends to increase with raising gCD.
Unusual phenomenon was observed at pH 4.3 where the
aggregation tended to decrease with increasing gCD

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C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

Fig. 5. The effect of excipients in 020% (w/v) gCD aqueous solution (pH 7.2  0.05)
on phase-solubility of irbesartan containing individual additives or all excipients in
% (w/v); 0.1% EDTA (^); 0.02% BAC (&); 0.1% EDTA + 0.02% BAC + 0.25% HPMC (D);
0.1% tyloxapol (); eye drop vehicle (~). The effect of individual or all excipients
signicantly increased the amount of dissolved drug; P < 0.05, one-way ANOVA
followed by Turkeys test.

concentrations, a phenomenon that can possibly be related to the

shift of pKa2 up on complexation with gCD. The ability of drug
molecule to interact with surrounding complexes is one of the
factors governing the aggregate formation, i.e., ionized irbesartan
molecules have less tendency to interact with gCD molecules
(K1:1 = 230 M1) than the unionized molecules (K1:1 = 450 M1 at
pH 4.3). The results show that the ionized drug molecules have
greater tendency to form aggregates although they have less
afnity for inclusion in hydrophobic gCD cavity. Previous studies
have shown that the aggregation is related to the CE, i.e., the actual
concentration of complexes, and not to the K1:1 value, i.e., the
afnity for the CD cavity (Messner et al., 2011b,c).
3.4. Effect of excipients on solubility and permeation
The effect of excipients on phase-solubility proles is displayed
in Fig. 5 and the stability constants and CE values are listed in
Table 3. The excipients chosen and their concentrations were
determined based on their use in marketed eye drop products or
based on our previous use of a given excipient. In general, the
recommended concentration for BAC in eye drop formulation is
0.010.02% (w/v), and 0.1% (w/v) EDTA is commonly used in
combination BAC to enhance its antibacterial activity (Richards
and McBride, 1973; Ryan et al., 2011). The effects of HPMC and
tyloxapol concentration on the gCD solubilization of irbesartan
were previously tested to determine their appropriate amount in
the eye drop vehicle, at concentration higher than 0.25% (w/v) of
HPMC and 0.1% (w/v) tyloxapol, dissolved drug amount is slightly
increase. According to the ratio of CE, BAC does not enhanced the
solubilizing effect of gCD, possibly due to the competition between
drug and BAC for forming complex with CD (Loftsson et al., 2003)
whereas EDTA, HPMC and tyloxapol were shown to increase in the
solubility. The increase in gCD solubilization of irbesartan by EDTA
and HPMC could be due to electrostatic forces and hydrogen
bonding, respectively (Loftsson and Frioriksdottir, 1998; Loh et al.,
2014). The polymerCD interaction on the solubilizing abilities of
CDs and drug availability have been observed and described
elsewhere (Faucci and Mura, 2001; Hirlekar et al., 2009; Loftsson
and Frioriksdottir, 1998; Ribeiro et al., 2003). HPMC, a long chain
water-soluble polymer with numerous hydroxyl groups, forms

Fig. 6. The effect of gCD concentration on the ux of irbesartan (A) and the
apparent permeation coefcients through semi-permeable membrane MWCO
12-14,000 (B) at pH 4.3  0.05 (&) and 7.2  0.05 (^). The pH had signicant effect
on the drug ux, but had insignicant on the permeation coefcients; P < 0.05,
student t-test.

hydrogen bonds with the peripheral hydroxyl groups of the CD

molecules and, thus, it is able to increase the amount of dissolved
gCD and gCD complexes in aqueous solutions. Consequently, the
drug/gCD complex formation is enhanced in the presence of
HPMC. Long chain water-soluble polymers are also known to
stabilize aggregates in aqueous solutions. Tyloxapol, non-ionic
surfactant oligomer, might improve the solubility of drug by either
improving drug wettability or micellar incorporation of drug and
drug/gCD complexes (Ba et al., 2008; Valente and Soderman,
2014). The greatest increase in irbesartan solubility is obtained in
eye drop vehicle containing 0.02% BAC, 0.1% EDTA, 0.25% HPMC,
0.1% tyloxapol, and 10% gCD, all in % (w/v). This could be attributed
to ternary complex formation in eye drop medium, enhance
complex efciency and solubilization of the drug/gCD complex.
The observed enhanced CE leads to enhanced irbesartan ux
through the MWCO 12-14,000 semi-permeable cellophane membrane. The plot of irbesartan ux in Fig. 5 shows how the
permeability coefcient is affected by the excipients in eye drop
formulation. Furthermore, the ux proles deviation from linearity
due to self-assemble of irbesartan/gCD complexes (Jansook and
Loftsson, 2009; Jansook et al., 2010a).
The molecular weight the irbesartan/gCD (1:1) complex
(
1725 Da) is less than the pore size of the membrane (MWCO

C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

87

Fig. 7. The fraction of aggregate complexes (fA) in irbesartan/gCD solution at pH 4.3  0.05 (A) and pH 7.2  0.05 (B) that are unable to permeate through semi-permeable
membrane MWCO 3500 Da, 60008000 Da, and 12,00014,000 Da.

12-14,000 Da) and, thus, aggregates containing up to eight

irbesartan/gCD (1:1) complexes can permeate the membrane.
Table 4 shows the size distribution data of irbesartan/gCD
complexes in aqueous solution containing the various excipients.
Small, medium, and large size aggregates were assigned based on
Gonzales-Gaitano et al. studies (Gonzalez-Gaitano et al., 2003; Wu
et al., 2006). The particle diameter varies from 1 nm to 3 mm.
Aggregates are present in the aqueous vehicles but their mass
distribution are very low. Additional observation of surface activity
of irbesartan/gCD complexes in aqueous solution was obtained by
surface tension measurements. The complex formation decreased

the surface tension by about 14 mN/n compared to pure water and

pure aqueous gCD solutions. Addition of the excipients resulted in
further reduction of the surface tension by about 33, 26, and
34 mN/m for BAC, HPMC, and tyloxapol, respectively. EDTA did,
however, not affect the surface tension.
The role of excipients on the irbesartan release (Fig. 8) can be
ascribed to their molecular structure and physiochemical properties. Addition of tyloxapol to the aqueous gCD media resulted in
the highest drug ux and this enhancement might be associated
with its molecular structural that is the interaction of the
irbesartan molecules with the amphiphilic moieties of the

Table 3
Effect of gCD and excipients on the apparent stability constant (K1:1) and the complexation coefcient (CE) of irbesartan (n = 34) at pH 7.2  0.05.
Excipients

K1:1 (M1)

CE

Ratioa

gCDb
gCD + 0.1% EDTA
gCD + 0.02% BAC
gCD + 0.25% HPMC
gCD + 0.1% Tyloxapol
gCD + All excipients

230
510
200
440
3900
5600

0.06
0.12
0.05
0.11
1.06
1.54

1
2
0.83
1.83
17.7
25.7

a
b

The ratio is calculated by dividing CE of excipients by CE of reference medium.

Reference medium (10% (w/v) gCD).

88

C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

Table 4
The DLS results of IBE/gCD complexes size analysis in aqueous solution (pH
7.2  0.05) containing individual excipients or all excipients, data reported as
hydrodynamic diameter (d) in nano-scale range, intensity area (%A) and, mass
distribution (%M). The samples were ltered through 0.45 mm membrane lter
before particle size determination.
Peakb

d (nm)

%A

%M

No additive

1
3
4

1
600
1500

22
76
2

99.99

0.1% EDTA

1
3
4

1
320
3100

13
85
2

99.99

0.02% BAC

1
2
3
4

1
78.96
280
1700

49
8
40
3

99.99

Tyloxapol

1
3

1
270

24
76

99.29
0.71

HPMC

1
2
3
4

1
38
270
3100

24
7
18
51

99.99

All excipients

1
2
3

1
170
630

33.22
25.57
41.21

99.99

Excipients
a

Irbesartan in aqueous gCD 10% (w/v) solution.

Small (1), medium (23), and large (4) of size distribution in nano-scale range
(nm).
a

surfactant, and the equilibrium of free irbesartan molecules with

irbesartan molecules bound to tyloxapol and/or gCD. Tyloxapol
can be referred as gemini surfactant made up of hydrophobic
chains and hydrophilic head groups. It is possible that tyloxapol
forms complexes by interacting with the hydrophilic gCD surface
while the hydrophobic part of the tyloxapol molecule forms a noncovalent bonding with lipophilic drug molecules (Pineiro et al.,
2007; Valente and Soderman, 2014; Veiga and Ahsan, 2000).
Moreover, tyloxapol has much lower critical micelle concentration
(CMC) and lower limiting surface tension than the conventional
surfactants, the free molecules might tend to form micelles in
solution and the drug molecules might be incorporated into the
micelles. Consequently, the availability of drug in the aqueous
complexation media increases in the presence of tyloxapol. The
micelle sizes of tyloxapol has previously been investigated by
Regev and Zana (Regev and Zana, 1999) and their size found to be
smaller than micelles of monomer surfactants. In summary, the
small micelles and the surface tension lowering action of tyloxapol
contribute towards higher irbesartan ux through the semipermeable cellophane membrane. In case of HPMC, the hydrophilic
polymer has been known to enhance drug availability by enfolding
drug molecules into the hydrated polymer network and promote
complexing formation of drug/CD complexes (Loh et al., 2014;
Teixeira et al., 2014). However, its polymeric structure resulted in
increased viscosity of the aqueous complexation media and, thus,
retarded drug mobility from network polymer. In comparison to
aqueous solution containing tyloxapol, HPMC demonstrates less
surface tension lowering effect and results in large particle size
than tyloxapol. This decreases drug release from aqueous media
containing HPMC. It is obvious that the small amount of HPMC and
tyloxapol, in combination with the other eye drop components,
provide for a synergistic effect on solubilization and complexation
efciency of the drug/gCD complex. In addition to decrease surface
tension of a vehicle, these excipients tend to stabilize the

Fig. 8. The ux of irbesartan (A) and the apparent permeation coefcients (B)
through semi-permeable membrane with MWCO 12-14,000; 0.1% EDTA (&); 0.02%
BAC (D); 0.25% HPMC (^); 0.1% tyloxapol (); eye drop vehicle (), all in (% w/v).
The excipients, both alone and in combination, had signicantly effect on the drug
ux but insignicant effect on the permeation coefcients; P < 0.05, one-way
ANOVA followed by Turkeys test.

aggregates, the nano- or microparticles in the aqueous eye drop

formulation.
4. Conclusion
Formation of water-soluble irbesartan/gCD inclusion complexes has remarkable effect on the irbesartan solubility and
availability. Optimization of the medium pH (i.e., the irbesartan
ionization) has profound effect on the gCD solubilization,
complexation and drug availability in aqueous irbesartan/gCD
complex solutions, increasing with enhanced CE while changes in
K1:1 did not have notable effect. FT-IR, 1H NMR, and XRPD
conrmed formation of inclusion complex and TEM analysis
showed evidence of irbesartan/gCD complex aggregates. Presence
of excipients in the aqueous gCD solution or eye drop medium was
shown to have signicant effect on formation of irbesartan/gCD
inclusion complexes and drug availability in the aqueous gCD
media. The study shows that good understanding of the various
drugexcipient and excipientexcipient interactions as well as
careful formulation optimization are essential for successful
formulation development.

C. Muankaew et al. / International Journal of Pharmaceutics 474 (2014) 8090

Acknowledgements
The study was gratefully supported by the grants from Icelandic
Center of Research, (RANNS), and University of Iceland. The
authors are gratefully acknowledging the support of Scientic and
Technology Research Equipment Centre, Chulalongkorn University,
Bangkok, Thailand.
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