Spectroscopic characterization of both aqueous and solid-state diacerhein/hydroxypropyl-β-cyclodextrin inclusion complexes

Article abstract of DOI:10.1016/j.saa.2014.02.055

Diacerhein, a poorly water soluble antirheumatic prodrug, was spectroscopically characterized to form inclusion complexes with hydroxypropyl-β-cyclodextrin (HPβCD) in both aqueous solution and in solid phase. Complexation with the hydrophilic carriers was used to improve the solubility and dissolution rate of the compound. The kinetics of the prodrug degradation to the active rhein in aqueous buffer solution were also investigated as a function of HPβCD concentration. The solid complexes prepared by different methods such as physical mixture, kneading, co-evaporation method and freeze dried method in 1:1 M ratio, were characterized by DSC and FTIR. The dissolution profiles of solid complexes were determined and compared with diacerhein alone and their physical mixture, in the simulated intestinal fluid at 37 C. The accurate molecular spectroscopic characterization of diacerhein in the presence of different amounts of aqueous cyclodextrins was essential to determine the correct binding constants for the diacerhein/HPβCD system. The binding constants were also validated by UV spectrometry and HPLC procedure in order to compare the values from the different methods. Higuchi-Connors phase solubility method has proved not suitable when either the free or/and the complexed prodrug degrade in aqueous solution.

Full text of DOI:10.1016/j.saa.2014.02.055

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 355–360
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectroscopic characterization of both aqueous and solid-state
diacerhein/hydroxypropyl-b-cyclodextrin inclusion complexes
Stefania Petralito a,1, Iacopo Zanardi b,1, Romina Spera a, Adriana Memoli a, Valter Travagli b,
⇑
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Roma, Italy
b Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
Diacerhein (DARh) is a prodrug of the
IL-1 converting enzyme inhibitor
Rhein (Rh).
The interaction of DARh with HPbCD
has been studied by molecular
spectroscopy.
Both the free and the complexed
DARh can undergo hydrolysis to
generate Rh.
ꢀ
ꢀ
DARh and Rh have some coincident
spectroscopic parameters.
Accurate experimental and
spectroscopic settings are essential
parameters.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2013
Received in revised form 4 December 2013
Accepted 11 February 2014
Available online 28 February 2014
Diacerhein, a poorly water soluble antirheumatic prodrug, was spectroscopically characterized to form
inclusion complexes with hydroxypropyl-b-cyclodextrin (HPbCD) in both aqueous solution and in solid
phase. Complexation with the hydrophilic carriers was used to improve the solubility and dissolution rate
of the compound. The kinetics of the prodrug degradation to the active rhein in aqueous buffer solution
were also investigated as a function of HPbCD concentration. The solid complexes prepared by different
methods such as physical mixture, kneading, co-evaporation method and freeze dried method in 1:1 M
ratio, were characterized by DSC and FTIR. The dissolution profiles of solid complexes were determined
and compared with diacerhein alone and their physical mixture, in the simulated intestinal fluid at 37 °C.
The accurate molecular spectroscopic characterization of diacerhein in the presence of different amounts
of aqueous cyclodextrins was essential to determine the correct binding constants for the diacerhein/
HPbCD system. The binding constants were also validated by UV spectrometry and HPLC procedure in
order to compare the values from the different methods. Higuchi–Connors phase solubility method has
proved not suitable when either the free or/and the complexed prodrug degrade in aqueous solution.
Ó 2014 Elsevier B.V. All rights reserved.
Keywords:
Diacerhein
Cyclodextrins
Hydrolytic degradation
Binding constants
Molecular spectroscopy
Introduction
Diacerhein or diacetylrhein or diacerein (4,5-diacetyloxy-9,
10-dioxoanthracene-2-carboxylic acid, see Fig. 1) [1], hereinafter
referred to as DARh, is an anthraquinonic drug used for the
treatment of osteoarthritis, mainly as a prodrug after metabolic
transformation to the active Rhein (Rh) [2,3]. Recently, increasing
⇑
Corresponding author. Address: Dipartimento di Biotecnologie, Chimica
e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy.
Tel.: +39 0577234317; fax: +39 0577234254.
E-mail address: valter.travagli@unisi.it (V. Travagli).
Both the authors equally contributed to this work.
1
http://dx.doi.org/10.1016/j.saa.2014.02.055
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

356
S. Petralito et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 355–360
Samples were kept in the dark thermostatted at 37 0.5 °C and the
hydrolysis reaction of DARh to Rh was monitorated by UV–vis (for
both DARh and Rh) and fluorescence spectra (only for Rh) [11].
Absorption spectra were recorded on a Perkin Elmer UV–vis Lamb-
da 25 spectrophotometer at 344 nm, corresponding to the maxi-
mum absorption wavelength of DARh, using 1 cm quartz cell.
Fluorescence measurements were carried out by a Perkin–Elmer
LS50B fluorimeter using 1 cm quartz cell, slit width was 5, excita-
tion and fluorescence emission wavelength of 435 nm. In both
cases, a software was used for data storage and processing. Differ-
ent calibration curves, for each cyclodextrin concentration, were
used to calculate Rh formation and DARh disappearance in solu-
tion. The observed first-order rate constant (Kobs) for the degrada-
tion was obtained from a non linear regression analysis of [DARh]/
[DARh]0 (where [DARh] is the concentration at a given time t and
[DARh]0 is DARh initial concentration) plotted vs. time [12]. All
measurements were carried out at least in triplicate.
Fig. 1. Structural formula of DARh.
attention was devoted to the potential off-label therapeutic prop-
erties of DARh, including anti-oxidative, anti-inflammatory, anti-
leishmanial and in prevention of alcohol-induced liver diseases
[4–7]. DARh is practically insoluble in water at acidic and neutral
pH, while its solubility increases in alkali. The latter environment,
however, lead to a very fast hydrolytic degradation, with produc-
tion of Rh [8]. Because of the low water solubility, further pharma-
cological development and practical application of the DARh itself
are limited. However, the formation of cyclodextrin derivatives for
achieving an enhanced aqueous solubility is experiencing a grow-
ing interest [9,10]. In addition to the enhanced solubility, another
advantage for using DARh/cyclodextrin complex could rise from
the control of the hydrolytic reaction into Rh, limiting its side
effects and consequently improving the poor tolerability of oral
DARh therapy. However, the DARh/Rh conversion should be
adequately taken into account for a correct host–guest evaluation,
also in light of recent information about the solubility and spectro-
scopic properties of Rh/cyclodextrin inclusion complex [11]. There-
fore, a deepen investigation about the effect of cyclodextrins on the
hydrolysis rate of DARh in aqueous solution has been reputed of
particular interest. The aim of this work was to study the effect
of hydroxypropyl-b-cyclodextrin (HPbCD) complexation on the
solubility and hydrolytic pattern of DARh by both spectroscopic
and chromatographic techniques. Moreover, for a comprehensive
characterization, solid-state complexes were prepared by freeze-
drying, co-evaporation, and kneading methods to confirm the oc-
curred complexation and to verify the influence of preparation
technique on improvement of DARh/HPbCD complex dissolution
in aqueous media. Selective physicochemical determinations
based on differential scanning calorimetry (DSC) and Fourier
Transformed-IR (FT-IR) were also investigated.
Stability constant determinations
Spectroscopic studies. The concentration of DARh in buffered
solution pH 7.2 both in the presence and in the absence of HPbCD,
was measured by absorbance (see above). Different calibration
curves, for each cyclodextrin concentration, were used to calculate
DARh concentration. Spectroscopic properties of DARh in the
presence of HPbCD were also used to calculate association constant
KIst from UV–vis data by using a linear curve fitting procedure (Eq.
(1)) [13].
ꢀ
ꢁ
e0
þ
e1 ꢂ KIst ꢂ ½HPbCDꢃ ꢂ ½DARhꢃ
1 þ KIst ꢂ ½HPbCDꢃ
Abs ¼
ð1Þ
A 1:1 inclusion complex for DARh/HPbCD interaction was
assumed, because it is the most commonly claimed and usually
justified stoichiometric ratio for CD–drugs complexes [14], as well
as it had been also verified in the case of interaction between Rh/
CDs [11].
For all absorbance measurements, aliquots of fresh DARh stock
solution was poured into quartz cells of 10-mm path length
(capacity about 4 mL) and then HPbCD from 0 to 100 mM was
added drop wise into cuvette and diluted to the final volume and
desired concentration. The solutions were stirred for 15 min and
then immediately analyzed. All measurements were carried out
at least in triplicate.
Kinetic determination: hydrolysis reaction. In the systems in which
the 1:1 stoichiometry is assumed, it is possible to use the influence
of the cyclodextrin in the hydrolysis rate of the compound to
II
st
calculate the complex stability, K with the kinetic method
Materials and methods
[15,16] and Eq. (2) can be used:
ꢂ
ꢃꢂ
ꢃ
D
D
Kobs
kc
½DARhꢃ
DKobs
Dkc
II
st
Materials
D
Kobs ¼ K 1 ꢁ
½HPbCDꢃ ꢁ
kc
ð2Þ
DARh (lot no. F07198901.0.01) was provided by TRB Chemedica
(Switzerland) and HPbCD (KleptoseÒ HP, MW = 1400, degree of
molar substitution 0.75–0.95, batch number 813447) was the gen-
erous gift from Roquette (France). All other materials and solvents
used were of analytical grade or purer. Freshly distilled water was
used throughout the experiments.
where
D
Kobs = k0 ꢁ Kobs and
D
kc = k0 ꢁ kc, with k0 representing the
hydrolysis rate constant for the non-catalyzed reaction (i.e. in the
absence of HPbCD), kc the hydrolysis rate constant of the guest in
the form of the inclusion complex and Kobs the experimental hydro-
lysis rate constant determined at the different HPbCD concentra-
II
st
tions. The K and kc values were obtained by non-linear fitting of
the Kobs data using the Gnuplot software package 4.0 [17].
In solution studies
HPLC-method. The chromatographic experiments were carried out
for the determination of the apparent association constant by using
a Perkin Elmer series 200 LC controller Pump and a Perkin Elmer
series 200 UV/vis detector (detection: 344 nm for DARh). A re-
Hydrolytic degradation studies
A stock solution of DARh (10ꢁ3 M) was prepared in DMSO. A
volume of 2.5 mL of the stock solution was added to the pH
7.2 0.1 phosphate buffered saline (PBS) containing HPbCD
(range 0–100 mM) to make a final volume of 25 mL (10ꢁ4 M DARh).
versed phase column Chromasyl 100 C18 5
lm 250 ꢂ 4.6 mm
(Higgins Analytical) was used with methanol–water (30/70 v/v),

S. Petralito et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 355–360
357
in which HPbCD was dissolved at different concentrations
(0–25 mM) as mobile phase (flow rate: 0.7 mL/min; elution time:
15 min). The chromatographic experiments were carried out at
22 0.5 °C. The DARh concentration in the injected solution was
for absorbance 4000–400 cmꢁ1; the resolution was 2 cmꢁ1 and the
spectra used were the result of averaging 100 scans.
Dissolution studies
10ꢁ4 M and the injection volume was 20
lL in all experiments.
The dissolution profile of DARh alone, PM and of its inclusion
complexes were studied using USP XXIII dissolution test for solid
oral dosage forms test with a paddle stirrer (apparatus II type).
The sample, corresponding to 50 mg of the prodrug (alone or in
its inclusion complex) were placed into 1000 mL of pH 7.2 0.1
phosphate buffer used as dissolution medium to simulate intesti-
nal medium. The stirring speed of the paddle was 100 rpm, and
the temperature was maintained at 37 0.5 °C. The samples were
withdrawn at regular time intervals, filtered through Whatman
polycarbonate filter paper and analyzed by UV spectrophotometer
at 344 nm to spectrophotometrically determine the amount of
DARh dissolved. The volume of each sample taken out was re-
placed by fresh dissolution medium and the actual DARh concen-
tration was corrected. The dissolution experiments were
conducted, at least in triplicate, for a time equal to 1 h, based on
preliminary experiments showing that in these experimental con-
ditions, a significant hydrolysis of DARh to Rh at pH 7.2 would take
place only after 60 min.
All measurements were carried out at least in triplicate. The reten-
tion behavior of DARh was dependent by the drug partition coeffi-
cients between the mobile and stationary phases. In the presence
of cyclodextrins, there is an additional contribution in the elution
behavior due to the complexation process and the capacity factors
for DARh were monitored in the presence of increasing concentra-
III
st
tion of HPbCD. The apparent stability constant of the complex, K
,
was determined using Eq. (3) [18]:
x
III
st
1
1
K
½HPbCDꢃ
¼
þ
ð3Þ
k0 k0s
ks0
where k0 is the capacity factor at each cyclodextrin concentration,
[HPbCD], k0s is the solute capacity factor in the absence of cyclodex-
trin, and the exponent x is the stoichiometry coefficient. For a 1:1
stoichiometry complex, a plot of 1/k0 vs. [HPbCD] yields a straight
line and K is obtained from the slope-to-intercept ratio.
Solid systems
Results and discussion
Sample preparation
Solid inclusion complexes of DARh and HPbCD were prepared in
1:1 M ratios by: physical mixture (PM), kneading method (KN), co-
evaporation (COE) and freeze-drying technique (FD). DARh and
HPbCD were triturated in mortar with small volume of a solvent
mixture of water:ethanol to obtain a kneading binary system.
The thick slurry was kneaded for 60 min and then dried at 45 °C
until constant weight. The dried mass was pulverized and sieved
through mesh #100. Co-evaporated inclusion complex of DARh
and HPbCD in 1:1 M ratio was prepared by dissolving the prodrug
in a solvent such as methanol and the cyclodextrin in water, sepa-
rately. The latter was then added to the former and stirred to
achieve equilibrium. The resulting volume was filtered through
In solution studies
Hydrolytic degradation studies
As can be seen from Fig. 2, the UV–vis spectra of 10ꢁ5 M DARh
and Rh solutions are different for the absorption maximum at
about 345 nm and 435 nm, respectively. On the contrary, they have
in common the absorption maximum around 260 nm. Moreover, it
is useful to recall that only Rh emits fluorescence if excited at
435 nm.
Thus, by following spectrophotometrically both the decrease of
DARh peak at 343 nm and the increase of Rh peak at 435 nm vs.
time, as well as fluorimetrically the appearance of the two emis-
sion fluorescent peaks at 513 nm and 580 nm due to the Rh forma-
tion with excitation at 435 nm, it is possible to study the hydrolysis
reaction [11]. In fact, DARh is decomposed at basic and acid pH val-
ues to Rh. The hydrolytic degradation studies, performed at neutral
pH, evidenced such a temporal pattern of DARh hydrolysis, at the
experimental conditions adopted (Fig. 3). Moreover, the influence
of HPbCD (0–100 mM) on the hydrolytic degradation of DARh at
pH 7.2 was investigated. As previously stated, the first-order ki-
netic degradation of DARh can be expressed either in a direct man-
ner by the decrease of concentration of DARh with time
0.45 lm filter and then dried by a rotary evaporator (Buchi Rotava-
por-KRvr 65/45). Freeze-drying inclusion complex of DARh and
HPbCD in 1:1 M ratio was prepared by dissolving the drug in aque-
ous solution of HPbCD. The suspension was stirred and then fil-
tered through 0.45 lm filter, freezed in N2 liquid and lyophilized
by a LIO5P apparatus (Cinquepascal, Milan, Italy) equipped with
an Edwards RV5 vacuum pump, consecutively. The physical mix-
ture (PM) of DARh and HPbCD in 1:1 M ratio was prepared by indi-
vidually mixing the components.
Characterization
DSC (thermal analysis)
Differential scanning calorimetry (DSC) measurements of the
pure materials (DARh and HPbCD), of the binary systems and PM
were carried out using a calorimeter (Setaram Instrumentation,
Caluire, France), model DSC 131. The thermal behavior was studied
by heating all accurately weighed samples (6 mg) in a pierced alu-
minum crucible from 50 to 350 °C. All measurements are made un-
der a nitrogen flow of 20 cm3/min at the scan rate of 10 °C/min
using an empty pan as reference. Indium (99.98%, mp 156.65 °C)
was used as standard for calibrating the temperature.
FT-IR
The infrared spectra were obtained with a Fourier transform
infrared (FTIR) spectrophotometer (Paragon 1000 FT-IR, Perkin El-
mer). The samples of pure drug, CD, PM and inclusion complexes,
were prepared by the potassium bromide disc method and scanned
Fig. 2. Full UV–vis absorption spectra of Rh and DARh.

358
S. Petralito et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 355–360
Fig. 4. Scheme of DARh/HPbCD complex formation in the presence of products
derived from hydrolysis (MARh: monoacetylrhein; asee Ref. [11]).
Fig. 3. UV–vis spectra of DARh vs. time: decrease of the absorbance of DARh at the
the guest one (DARh 10ꢁ4 M) is about 45 Mꢁ1, resulting from the
linear slope-to-intercept ratio (R2 = 0.993). On the other hand, the
same linear relationship validates the 1:1 stoichiometry of the
inclusion complex. In such a condition, the Benesi–Hildebrand
method [21] can also be applied to calculate the equilibrium con-
stant of DARh/HPbCD complex. Evidence for the 1:1 complex for-
mation was obtained by using the double reciprocal plot of
Benesi–Hildebrand equation (see supplementary information as
Fig. S1). For completeness’ sake, in the presence of HPbCD a little
absorption enhancement and a little bathocromic shift for the
maximum at 344 nm have been observed (data not shown).
maximum absorption wavelength (kmax = 343 nm) and appearance of
corresponding to Rh formation (kmax = 434 nm).
a peak
t
ð½DARhꢃ ¼ ½DARhꢃ0eꢁK Þ or indirectly by using Rh spectroscopic
obs
data obtained by UV–vis spectroscopy and by fluorimetry of Rh
t
according to the relation ½Rhꢃ ¼ ½Rhꢃ0ð1 ꢁ eꢁK Þ, where [Rh]0 is
obs
the final Rh concentration corresponding to the initial concentra-
tion of DARh, [Rh] is the concentration of Rh at the t time degrada-
tion and Kobs is the first-order degradation rate constant. Fitting of
[Rh]/[Rh]0 values against t leads to the degradation rate constant
values. Kobs values obtained by UV–vis measurements of DARh
and by UV–vis and fluorescence measurements of Rh are substan-
tially in agreement with each other. When no CD was present in
solution the rate constant for the hydrolysis was 0.23 hꢁ1, while
at 100 mM HPbCD an increase in stability more than 50% was ob-
served (Kobs = 0.11 hꢁ1). A stabilizing effect on DARh hydrolysis
reaction with increasing HPbCD concentration appears evident.
Such a stabilization can be explained according with the inclusion
of DARh molecule in the cyclodextrin cavity. Specifically, the
hydrolysis kinetic follows the so-called ‘‘saturation behavior’’, con-
sistent with 1:1 binding between DARh and HPbCD (Table 1). In de-
tail, the rate of reaction changes with the increase of HPbCD
concentration and the Kobs does not linearly relate to the increasing
concentration of added HPbCD, but rather asymptotically ap-
proaches a minimum value [19,20]. Such a trend can be explained
by the model of Fig. 4: when DARh is in solution, it comes into
equilibrium with both HPbCD, to give the inclusion complex, and
the hydrolysis products (monoacetylrheinate I and II, referred as
MARh, and Rh), which in turn compete in the inclusion process
with the HPbCD. Under the experimental conditions adopted, the
DARh/HPbCD complex formation proves to be faster than hydroly-
sis processes.
Kinetic determination: hydrolysis reaction. The determination of the
stability constant of the complex DARh/HPbCD can be obtained by
the kinetic method [12]. In order to calculate the degradation rate
constant Kc for DARh within the cyclodextrin cavity, the data for
HPbCD was fitted to the Eq. (2). The same Kc value of 0.07 hꢁ1
was obtained both from absorption and fluorescence data. Such
II
st
an equation can be also used to get the stability constant, K , of
the DARh/HPbCD complex from the same absorption and fluores-
cence data and Kst values near to 50 Mꢁ1 and 75 Mꢁ1 were ob-
tained, respectively. The agreement between the experimental
data and the model is quite good, confirming both the formation
of 1:1 complex and the proposed degradation scheme (Fig. 4).
HPLC method. The retention factors of DARh in the experimental
condition adopted were weakly decreased with increasing concen-
tration of HPbCD, according to the coexistence of two physico-
chemical processes: the solute complexation by HPbCD, and the
transfer of uncomplexed solute from the hydro-organic phase to
the stationary phase. In these conditions, the interaction of the
DARh/HPbCD with the stationary phase can be considered as neg-
ligible [22], so it is not taken into account and the retention factors,
k, for DARh can be derived from Eq. (3). The linear relationship be-
tween 1/k and HPbCD concentration, with correlation coefficients
higher than 0.988 (data not shown), indicates that the behavior
of these equilibria is well described by the model assuming a 1:1
Stability constant determinations
Spectroscopic studies. The UV–vis spectra of DARh in the presence
of various HPbCD concentrations were also used to calculate the
association constant, KsIt (Eq. (1)). The value of KsIt in the presence
of HPbCD concentration (25–100 mM) in excess with respect to
stoichiometry of host–guest interactions. The apparent stability
III
st
constant K has been calculated from the linear slope-to-intercept
ratio, resulting equal to 22 Mꢁ1
.
In relation to the aqueous phase investigation, the evaluation of
Kst 1:1 apparent stability constants for DARh/HPbCD complex car-
ried out with different method leads to approximately comparable
values: UV–vis spectrophotometry: 45 Mꢁ1, kinetic determination:
either 50 Mꢁ1 or 75 Mꢁ1, depending on the spectroscopic method,
and HPLC: 22 Mꢁ1. In particular, the latter one shows how impor-
tant it is to identify the chemical species that are in competition
with the formation of the inclusion compounds. In fact, the values
achieved by our studies are noticeably different from the DARh/
HPbCD Kst 1:1 value of 194 Mꢁ1, previously obtained by phase sol-
ubility studies [9]. On the other hand, such an extent is close to the
Table 1
Observed first-order rate constant (Kobs) values for the DARh degradation based on
the appearance of Rh.
HPbCD
concentration
(mM)
Kobs (minꢁ1) from UV–
vis spectra
Kobs (minꢁ1) from
fluorescence spectra
0
25
50
75
100
0.2315 0.0058
0.1444 0.0063
0.1158 0.0043
0.0966 0.0038
0.1068 0.0053
0.2285 0.0135
0.1491 0.0076
0.1379 0.0068
0.1011 0.0023
0.1134 0.0068

S. Petralito et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 355–360
359
values (240, 243 and 350 Mꢁ1) estimated for Rh/HPbCD complex
by a variety of methods [11]. This outcome can be basically justi-
fied taking into account that for a right application of the phase sol-
ubility method, the equilibrium phase must be fully achieved
requiring generally long experimental times (more than a week)
and therefore the complete stability of the guest molecules during
the analysis must be assured. In the phase solubility methods, the
presence of HPbCD makes this process slower but the value of Kst
calculated in these conditions results in any case wrong due to
the transformation of DARh in Rh. Moreover, it is noteworthy that
if only the absorbance of maximum in the UV region is considered,
the hydrolytic degradation of DARh–Rh cannot be evidenced
(Fig. 2) and the obtained inclusion parameters can be consequently
inaccurate. Such erroneous experimental conditions have been re-
ported in recent studies that have been previously cited, where the
DARh inclusion parameters in cyclodextrins were evaluated at
258 nm [9,10].
Solid systems
Thermal analysis
The DSC diagrams of the binary system are compared with those
of the starting compounds (DSC diagrams are shown in the supple-
mentary information as Fig. S2). In such analysis, the disappearance
of melting peak of the guest molecule in the binary system is con-
sidered to be the evidence of the occurred complexation; besides,
the decrease of the endothermic peak associated to HPbCD dehy-
dratation in comparison with that of the pure HPbCD as well as
the shift of melting peak of the guest molecule are indicative of
an interaction of the drug with the cyclodextrins. As it is possible
to note, the curve referred to pure cyclodextrin presents a broad
endothermic peak in the interval 79–116 °C (90 °C) that may be
attributed to the loss of water; all binary systems show a decrease
in this dehydration endothermic peak, observed in the
DH value,
Fig. 5. Comparison of FT-IR spectra (transmittance, T%) of pure DARh, HPbCD, as
well as solid inclusion complexes in 1:1 M ratios by physical mixture (PM),
kneading method (KN), co-evaporation (COE) and freeze-drying technique (FD).
indicating an interaction drug/HPbCD. As far as curve of binary sys-
tems obtained with physical mixture and kneading are concerned, a
partial reduction of dehydration peak of HPbCD and only a decrease
and a shift toward higher temperature of the melting point of the
pure DARh are detected. This is a proof of the presence of the pure
DARh in the PM and in the KN products that has not completely
interacted within the CD. Finally the disappearance of endothermic
peak of pure DARh (at onset 232 °C) is observed in the diagrams re-
lated to the binary systems obtained with the FD and COE methods,
it is confirmed the strong interaction between CD and host investi-
gated molecules.
Dissolution studies
Dissolution studies have been done to study the effect of solid
state complexation. The resulting dissolution profiles are given in
the supplementary information (Fig. S3). All the prepared binary
systems exhibit a drug dissolution rate faster than that of the pure
DARh, with a dissolution time scale of a few minutes. In detail, the
DARh dissolution % value at 5 min with respect to that of DARh
amount was found to be approximately 100, 93.9, 72.29 and
51.98 for FD, CE, KN and PM, respectively. Furthermore, in the case
of pure drug, only 55% of DARh was dissolved even after 60 min.
The significant improvement in dissolution characteristics of the
complexes was attributed to reduced interfacial tension between
the solid particles of DARh and the dissolution medium, leading
to greater rate of dissolution. The increase in the dissolution rate
of DARh physically mixed with CD was possibly due to local solu-
bilization operating in the micro environment or the hydrody-
namic layer surrounding the drug particles as reported by
Becirevic-Lacan et al. [23]. In situ inclusion process might have re-
sulted in increased amount of dissolved drug in case of physical
mixtures [24]. It is worth noting that during the dissolution tests,
no significant hydrolysis was detected while, if the tests are pro-
longed for more than 60 min, a significant Rh concentration ap-
pears in the dissolution medium.
FT-IR spectroscopy
The interaction between host and guest molecule was effec-
tively characterized by FTIR spectroscopy (Fig. 5). The principal
absorption peaks of DARh were observed at 3300 cmꢁ1 (OAH,
stretch, broad, COOH), 3069 cm–1 (CAH, stretch, aromatic),
2935 cmꢁ1 (CAH, stretch, aliphatic, sym), 1770 cmꢁ1 (C@O, stretch,
ester), 1693 cmꢁ1 (C@O, stretch, ketone), 1679 cmꢁ1 (C@O, stretch,
COOH), 1593 cmꢁ1 (C@C, stretch, aromatic), 1450 cmꢁ1 (CAO,
stretch, COOH), 1026 cmꢁ1 (CAO, stretch, ester), 760 cmꢁ1 (m
substituted benzene), and 704 cmꢁ1 (benzene).
While spectra of products obtained with physical mixtures and
kneading showed approximately a superposition of DARh and
HPbCD spectra, the intensity and the shape of bands in the region
1770 cmꢁ1 and 704 cmꢁ1 in the spectra of binary systems obtained
with the FD and COE methods changed and band assigned to car-
boxyl carbonyl stretching disappears; disappearance of carbonyl
absorption disappearance of carbonyl absorption band assigned
to carboxyl carbonyl stretching suggesting formation of H-bonds
between carbonyl group of DARh and hydroxyl group of host
cavity.
Conclusion
The use of HPbCD allows to DARh both to improve the solubil-
ity characteristics as well as to increase its hydrolytic resistance.

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S. Petralito et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 127 (2014) 355–360
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Furthermore, as expected, also the method of preparation can
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