REV . CHIM. (Bucharest) 66 No. 1 2015 http:www.revistadechimie.ro 17Host – guest System of Zofenopril and Randomly [631783]

REV . CHIM. (Bucharest) ♦ 66 ♦ No. 1 ♦ 2015 http://www.revistadechimie.ro 17Host – guest System of Zofenopril and Randomly
Methylated βββββ – cyclodextrin
Preparation, characterization and solubility
LUCRETIA UDRESCU1,2*, ADRIANA FULIAS1, IONUT LEDETI1, GABRIELA VLASE3, PAUL BARVINSCHI4, LUDOVIC KURUNCZI1,2,
LAURA SBARCEA1
1 “Victor Babeș” University of Medicine and Pharmacy, Faculty of Pharmacy, 2 Eftimie Murgu Sq., 300041 Timișoara, Romania
2 Institute of Chemistry Timisoara of the Romanian Academy, 24 Mihai Viteazul, 300223 Timisoara, Romania
3 West University of Timisoara, Research Center for Thermal Analysis in Environmental Problems, 16 Pestalozzi, 300115
Timisoara, Romania
4 West University of Timi’oara, Faculty of Physics, 4 V asile Pârvan, 300223 Timisoara, Romania
Zofenopril calcium (ZOF) is one of the newest angiotensin converting enzyme (ACE) inhibitor, highly lipophilic
and with low water solubility. This research investigates the interaction between ZOF and randomly
methylated β-cyclodextrin (RAMEB), in order to prove the formation of an inclusion complex that has an
enhanced water solubility profile for ZOF. This paper reports, for the first time, the physicochemicalcharacterization and the solubility profile of an inclusion complex between ZOF and RAMEB, which is prepared
using the kneading method. Different spectroscopic techniques, namely powder X-ray diffraction, ATR FT-IR
spectroscopy, were applied in order to prove the formation of the ZOF/RAMEB inclusion complex, both inwater and in solid state. The complex formation is further backed by thermal analysis (TGA/DTG/HF). The
obtained results confirm that the physicochemical properties of the ZOF/RAMEB binary system, prepared
using the kneading method, are different in comparison both with the parent substances and the correspondingphysical mixture, thus suggesting that an inclusion complex was formed. After the formation of the inclusion
complex with RAMEB, the solubility test has indicated that the water solubility of ZOF was increased by 4
times.
Keywords: zofeopril calcium, ATR-FTIR, PXRD, thermal analysis, solubility
* email: [anonimizat] calcium (ZOF), chemically named calcium
(2S,4S)-1-[(2S)-3-benzoylsulfanyl-2-methylpropanoyl]-4-phenylsulfanylpyrrolidine-2-carboxylate , is a sulfhydryl-
containing ACE inhibitor, as presented in figure 1a. Based
on its selective cardiac ACE inhibition, ZOF is an effectiveantihypertensive agent and a therapeutic option in
congestive heart failure and in acute myocardial infarction
[1-3]. ZOF is an ester prodrug of zofenoprilat (fig. 1b), whichis the bioactive acid form containing a sulfhydryl group [4].
The presence of sulphur in the ZOF molecule is responsible
for its remarkable antioxidant and cardio protectiveproperties [5, 6]. ZOF exhibits high lipophilicity (log P =
3.5) [1], while its major drawback is the low water solubility
(0.3 mg/ml reported by [1]), since this is the actual utilizedform in the oral formulations. Moreover, zofenopril calcium
exhibits the tendency of forming numerous polymorph
forms [7], thus resulting in decreasing its water solubility.The cyclodextrins are a class of cyclic oligosaccharides
consisting of six, seven or eight α-D-glucopyranose units,
named α-, β-, and γ-cyclodextrin, respectively. Their
particular hydrophobic cavity presents biomedical and
pharmaceutical interests, because they are able to forminclusion complexes with guest molecules ( i.e. drugs or
their lipophilic moieties) with an improved solubility,
stability or other physicochemical properties [8, 9]. Theattained amorphous solid state following the complex
formation using cyclodextrins [8, 10] has a potential benefit
for the oral formulations containing ZOF , due to theincreased water solubility.
Up to date, the studies on ZOF [11] and ZOF inclusion
complexes with native β-cyclodextrin [12] are scarce;
moreover, there is no scientific report on ZOF inclusion
complex with RAMEB. In this paper, the formation of the
inclusion complex between ZOF and RAMEB isinvestigated, as a consequence of ZOF molecularly
encapsulation within hydrophobic cyclodextrin cavity.
SO
CH3ONS
OO-
2Ca2+
HS
CH3ONS
OOH
a bFig. 1. The chemical structures of: (a) zofenopril
calcium and (b) zofenoprilat

http://www.revistadechimie.ro REV . CHIM. (Bucharest) ♦ 66 ♦ No. 1 ♦ 2015 18Therefore, we applied spectroscopic techniques and TGA/
DTG/HF thermal analysis as the most accurate and reliable
methods in the study of the host-guest interaction.
Experimental part
Apparatus
The spectrophotometric measurements were
performed by using a Spectronic Unicam-UV 300 UV-Vis
double beam spectrophotometer, with 1 cm matchedquartz cells.
X-ray diffraction studies of the pure substances (ZOF
and RAMEB) and of their binary systems (the correspondingphysical mixture and kneaded product) were performed
using a Bruker D8 Advance powder X-ray diffractometer,
in the range of 5-45° angular domain (2 θ), with CuK
radiation generated at 40 mA, 40 kV , and a Ni filter.
Thermal analysis was made by using a Perkin-Elmer
Diamond simultaneous TGA/DTA instrument. The DTAcurves (in μV) were changed with the heat flow (HF) curves
(in mW) in order to determine the heat effects. The thermal
behaviour of the substances was studied under an airatmosphere, at a flow rate of 100 mL/min and non-
isothermal conditions, by increasing the ambient
temperature up to 350 °C with a constant heating rate of10 °C/min.
The FTIR spectra were yielded using a Bruker Vertex 70
spectrometer equipped with a platinum ATR unit, typeBruker Diamond A225/Q. Each spectrum represents 64
co-added scans, at a resolution of 2 cm
-1, in the 4000-400
cm-1 wavenumber range.
Materials and reagents
Zofenopril calcium was a gift sample from Berlin-
Chemie Menarini (Berlin, Germany). Randomly methylated
β-cyclodextrin (average formula weight 1303.4, DS~12)
was purchased from Cyclolab R&D Ltd. (Budapest,
Hungary). The substances were used as received. All other
chemicals and reagents were of analytical grade. Allexperiments were performed using distilled water.
Binary systems preparation
The accurate weight of RAMEB for a 1:1 molar ratio
ZOF:RAMEB was triturated with an appropriate quantity of
water, at the ambient temperature, for 10 min, up tohomogenization. Then ZOF was slowly added to the paste.
The dissolution of the drug was assisted by adding small
quantities of water in the mixture. The paste was subjectto kneading for 1 h. The paste that was obtained by
performing this process was dried in the oven at 40°C, for
24 h. Then, the dried kneaded product, named ZOF/RAMEBKP , was pulverized and passed through a 75 μm size sieve.
The stoichiometric quantities of ZOF and RAMEB
corresponding to a 1:1 molar ratio were gently mixed in amortar, at the ambient temperature, for 10 min, in order to
get a homogenous blend, thus obtaining the corresponding
physical mixture at the same ratio as for the inclusioncomplex (ZOF/RAMEB PM). This binary system was used
for comparison with ZOF/RAMEB KP .
Calibration curve of ZOF and the solubility profile of the
kneaded product
A set of ZOF aqueous solutions was prepared, with
concentrations ranging between 9 –72 μg/mL. The
absorbance ( A) values, recorded at 248 nm, in UV , at 25 °C,
were represented as a function of ZOF concentration values(C in μg/mL), in order to achieve the calibration curve of
ZOF.The water solubility of ZOF within its inclusion complex
was determined as follows: an excess amount of kneaded
product was placed in 2 mL of distilled water, for obtaininga saturated solution. The mixture was shaken for 24 h, at
25°C, and then filtered on 0.45 μm cellulose acetate filter.
The clear supernatant was properly diluted and itsabsorbance was measured at 248 nm in UV , at 25 °C. The
residue dosing was performed by means of the calibration
ZOF curve.
Results and discussions
PXRD analysis
PXRD technique allows the observation of the crystalline
substance (i.e. ZOF) sharp peaks attenuation due to the
inclusion interaction with the amorphous cyclodextrin,RAMEB. The PXRD patterns of the native ZOF and RAMEB
and of their binary systems (the physical mixture and the
kneaded product) are presented in figure 2.
Fig. 2. PXRD patterns of the native substances (a), and their
corresponding binary systems (b)a
b
The PXRD spectrum of ZOF , presented in figure 2a,
emphasizes its highly crystalline nature by characteristicdiffraction peaks at 9.72, 13.74, 14.48, 16.00, 17.70, 18.42,
18.52, 19.00, 19.98, 20.56, 22.34, 24.60 2 θ degrees [13].
The PXRD pattern of RAMEB, as shown in figure 2a, hastwo broad peaks and many diffused peaks with low
intensities, which reveal its amorphous state. In figure 2b
is presented the PXRD spectrum of ZOF/RAMEB PM (fig.2b), which is almost an overlapping of the individual
diffraction spectra. By contrast, the ZOF/RAMEB KP
diffraction spectrum (fig. 2b) analysis reveals thediminished intensities of the ZOF characteristic sharp
peaks ( i.e. 16.00, 17.70, 19.00, 19.98, 21.93 2 θ). This result
emphasizes that the ZOF crystallinity is drastically reduced,due to the modifications in ZOF and RAMEB environment
following the inclusion complex formation process [14,
15].

REV . CHIM. (Bucharest) ♦ 66 ♦ No. 1 ♦ 2015 http://www.revistadechimie.ro 19Thermal analysis
Thermal analysis leads to valuable outcomes regarding
cyclodextrin inclusion complexes formation. The thermalbehaviour of the pure compounds and of their binary
systems is depicted in figure 3.
According to the HF curve (fig. 3a), the ZOF melting
point appears at 265 °C [13], this endothermic process
being followed by decomposition, a process that is
confirmed by the TGA curve. The RAMEB dehydration isrevealed by the HF curve (fig. 3b), which presents a broad
endothermic peak ranging between 40–140°C [16]. The
water loss is also shown by the TGA curve, in the sameinterval of temperature. The melting point of RAMEB
appears at a higher value than ZOF , as indicated by the
DTG curve.
The thermal behaviour of ZOF/RAMEB PM (fig. 3c)
reveals a diminished endothermic peak at 226°C, which
corresponds to the ZOF melting process. The endothermicpeak of ZOF melting process within ZOF/RAMEB KP is
reduced in comparison with that of the corresponding
physical mixture and it is shifted to a lower temperature,to 224°C (fig. 3d). Hence, a decrease in thermal stability of
the ZOF/RAMEB kneaded product is observed, as a
consequence of the amorphization of ZOF, as a guest
a b
c d
Fig. 3. The thermal profiles of ZOF (a), RAMEB (b), ZOF/RAMEB PM (c), and ZOF/RAMEB KP (d)
Fig. 4. ATR-FTIR spectra of: a) RAMEB, ZOF , ZOF/RAMEB physical mixture and ZOF/RAMEB kneaded product in 4000-2500 cm-1 spectral
region; b) ZOF and RAMEB in 2000-500 cm-1 spectral region; c) ZOF/RAMEB PM and ZOF/RAMEB KP in 2000-500 cm-1 spectral regionab c
compound, through the inclusion complex formation [17,
18].
ATR-FTIR analysis
The ATR-FTIR spectra of the pure ZOF and RAMEB, along
with their corresponding physical mixture and kneadedproduct are presented in figure 4.
The ATR-FTIR spectrum of pure ZOF (fig. 4a, 4b), is
characterized by the presence of peaks for the prolinegroup (at 1470 cm
-1) [19], the anti-symmetric and
symmetric stretching C=O vibrations at 1659 and 1612
cm-1, respectively. The Car–H stretching vibration arises at
3061 cm-1, the skeletal vibration of the aromatic ring appears
at 1582 cm-1, while the Car–H and Car–Car bending vibrations
appear at 772 and 746 cm-1, respectively [20]. The S-CH2stretching vibration is identified at 2876 cm-1 [19].
The FT-IR spectra of the inclusion complex prepared by
kneading, as presented in figures 4a and 4c, revealconsiderable differences in comparison with the
corresponding physical mixture and the pure compounds.
Almost all the characteristic peaks of ZOF are intact in thephysical mixture spectrum, but they are shifted (or even
absent) in the ZOF/RAMEB KP spectrum. By analyzing the
spectral data, we found that the ZOF C
ar–H stretching

http://www.revistadechimie.ro REV . CHIM. (Bucharest) ♦ 66 ♦ No. 1 ♦ 2015 20Fig. 5. a) UV absorption spectra of ZOF in distilled water, with concentrations ranging between 9 and 72 μg/mL; b) the calibration curve of
ZOF in distilled water.
vibration is shifted to 3055 cm-1 in ZOF/RAMEB PM, and it is
no more present in ZOF/RAMEB KP , thus indicating that
one of the two ZOF aromatic rings is entrapped within theRAMEB cavity. ZOF’s C=O stretching vibrations are still
present, although shifted in both binary systems. The proline
group vibration, which is identified at 1470 cm
-1 in the ZOF
spectrum, is shifted at 1466 cm-1 in ZOF/RAMEB PM, and
absent in the ZOF/RAMEB KP . At the same time, the ZOF
peak which corresponds to the S-CH2 stretching vibration
is absent from both binary systems, thus suggesting that
the inclusion complex was formed and the 4-
(phenylthio)pyrrolidyne is enclosed in the RAMEB cavity.
Solubility
The testing of the water solubility of our ZOF/RAMEB
inclusion complex is a valuable purpose of our
experimental approaches. Therefore, the saturation shake-
flask method [21, 22] was applied in order to measure theZOF water solubility, at 25 °C, as it is in ZOF/RAMEB KP ,
using the calibration curve of ZOF in water, at 25 °C (fig. 5).
The calibration curve of ZOF is characterized by equation
A = 0.0289· C + 0.0147 ( R = 0.9999), where A stands for
absorbance, measured at 248 nm, and C is ZOF
concentration in μg/mL.
The determination of the water solubility of the ZOF/
RAMEB KP was achieved by preparing a concentrated
solution, which was properly diluted [21, 22]. The UVspectrophotometric measurements have indicated that the
water solubility of the included ZOF is 1.199±0.014 mg/
mL, as an average value of five experimentaldeterminations. This result indicates that the water
solubility of ZOF is increased by four times in comparison
with the free ZOF (0.3 mg/mL), due to the solubilising effectof RAMEB.
The next step was to perform of a standard control
experiment. Therefore, a mass of 4.7 mg of ZOF/RAMEBKP , which is equivalent to 1.199 mg of ZOF , was dissolved
in distilled water and a clear solution was obtained. This
result indicates that the solubility of the ZOF/RAMEBinclusion complex is adequate for tablet dosage form [21,
22].
Conclusions
This paper reports for the first time the physico-chemical
characterization of an inclusion complex between ZOFand RAMEB. It also stresses the solubilising role of RAMEB
in improving the water solubility of the calcium salt of
zofenopril. By applying the kneading method, an inclusioncomplex between ZOF and RAMEB was formed, as proven
by ATR-FTIR spectroscopy, PXRD, and thermal analysis.
ab
The water solubility of ZOF/RAMEB KP was enhanced by
four times in comparison with pure ZOF.
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Manuscript received: 15.10.2014