Article

pubs.acs.org/molecularpharmaceutics

Molecular Dynamics, Physical Stability and Solubility Advantage

from Amorphous Indapamide Drug
Z. Wojnarowska,*,† K. Grzybowska,† L. Hawelek,†,‡ M. Dulski,† R. Wrzalik,† I. Gruszka,† and M. Paluch†
†
Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland
‡
Institute of Non-Ferrous Metals, ul. Sowinskiego 5, 44-100 Gliwice, Poland

K. Pienkowska and W. Sawicki

Department of Physical Chemistry, Medical University of Gdansk, Hallera 107, 80-416, Gdansk, Poland

P. Bujak
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00664 Warszawa, Poland

K. J. Paluch and L. Tajber

School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland

J. Markowski
ENT Department, Silesian Medical University, ul. Francuska 20, Katowice, Poland

ABSTRACT: This study for the first time investigates physicochemical properties
of amorphous indapamide drug (IND), which is a known diuretic agent commonly
used in the treatment of hypertension. The solid-state properties of the vitrified,
cryomilled and ball-milled IND samples were analyzed using X-ray powder
diffraction (XRD), mass spectrometry, nuclear magnetic resonance (NMR),
infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and
broadband dielectric spectroscopy (BDS). These analytical techniques enabled
us (i) to confirm the purity of obtained amorphous samples, (ii) to describe the
molecular mobility of IND in the liquid and glassy state, (iii) to determine the
parameters describing the liquid-glass transition i.e. Tg and dynamic fragility, (iv)
to test the chemical stability of amorphous IND in various temperature conditions
and finally (v) to confirm the long-term physical stability of the amorphous
samples. These studies were supplemented by density functional theory (DFT)
calculations and apparent solubility studies of the amorphous IND in 0.1 M HCl, phosphate buffer (pH = 6.8), and water (25 and 37 °C).
KEYWORDS: dielectric spectroscopy, molecular dynamics, glass transition, diuretic agents,
amorphous active pharmaceutical ingredients, tautomerization

■ INTRODUCTION
Indapamide (IND) is a sulfonamide diuretic drug used in the
During the past decade improvement of solubility of poorly
water-soluble medicines has become one of the most important
treatment of hypertension. Despite having a slightly different aspects of pharmaceutical research. In the literature one can
chemical structure than thiazides (e.g., hydrochlorothiazide), its find very few different approaches as to how to realize this task.
mechanism of action remains similar. It increases the urine volume One of the methods is to modify the active component by
by increasing the renal excretion of sodium, chlorine, potassium and means of salification. It means that the pure drug is transferred
magnesium ions. Apart from the diuretic action indapamide exerts into nitrate, hydrochloric or other salts which usually reveal
also spasmolytic effects on blood vessels, consequently reducing the
blood pressure.1 However, the commercial form of IND available on Received: February 28, 2013
the market works very weakly. The most probable reason is its low Revised: August 22, 2013
solubility (75 mg/L) and consequently poor bioavailability.2 Thus, Accepted: September 5, 2013
the question arises how to improve the solubility of indapamide? Published: September 5, 2013

© 2013 American Chemical Society 3612 dx.doi.org/10.1021/mp400116q | Mol. Pharmaceutics 2013, 10, 3612−3627
Molecular Pharmaceutics

■
Article

better solubility than the original compound.3,4 Additionally, EXPERIMENTAL SECTION

one can also apply several formulation strategies such as
Material. The tested indapamide hemihydrate 4-chloro-N-
micronization,5 molecular dispersion,6 incorporation of surfac-
tants, comilling with sugars,7 cyclodextrins or PVP and (2-methyl-2,3-dihydro-1H-indol-1-yl)-3-sulfamoylbenzamide
amorphization.8,9 The last method is a relatively simple (C16H16ClN3O3S·(1/2)H2O), MW = 365.83 g/mol with the
approach and thus widely employed to enhance the apparent chemical structure presented in Figure 1, was supplied from
solubility of pharmaceutical materials. As examples one can
mention amorphous verapamil hydrochloride,10 glipizide11 or
celecoxib.12 It should be noted that apart from affording a
higher apparent solubility (and consequently higher bioavail-
ability) an amorphous form can also improve processability of
drugs.13 For instance, by tableting an amorphous pharmaceu-
tical as opposed to the crystalline form one can reduce the
amount of additives in the tablet and thus simplify the
formulation. Unfortunately, preparation of amorphous drugs
may be also quite complex. First, an amorphous phase is
thermodynamically unstable, which in consequence may lead to
crystallization of the drug under storage conditions and
subsequent loss of the benefits arising from the amorphous Figure 1. Chemical structure of indapamide.
state.14 Second, as a result of higher chemical reactivity,
amorphous pharmaceuticals are prone to chemical degradation Quimica Sintetica (Madrid, Spain) as a white crystalline powder
as well as isomeric transformations during manufacturing and and was used without further purification. The powder X-ray
storage. Tautomerization, reversible proton migration in an diffractogram of crystalline IND (see blue line in Figure 2) was
organic molecule, is of a great importance in pharmaceutical
science since it has been shown that preparation of an
amorphous pharmaceutical, in which tautomerization reaction
takes place, can lead to a state which is chemically different
from its crystalline counterpart.15 This phenomenon can have
positive as well as negative impacts on the quality of amorphous
drugs. On the one hand, one can expect that the presence of
tautomerization products may affect the drug solubility, and
consequently it may enhance the bioavailability of the active
substance. Moreover, the occurrence of two or more tautomers
in the sample may reduce its crystallization tendency.34 Also,
tautomerization of drugs might lead to serious medical
consequences, since one cannot exclude that the biological
activity of the given tautomers may be different from that of the
parent drug. All these factors are reasons why in recent years
one of the main challenges of developing amorphous
pharmaceuticals is to understand the molecular interactions in
Figure 2. Thermal analysis of crystalline and amorphous forms of
the glassy and supercooled liquid phases and subsequently to IND. All DSC thermograms were obtained during heating at a rate of
predict the physical and chemical stability of amorphous drugs. 10 K/min. The inset panel shows the glass transition regions of
In this study we investigate the amorphous indapamide drug. quenched, cryomilled and ball-milled IND samples.
Polymorphic and pseudopolymorphic forms of indapamide16
have been identified and characterized, however no reports are
available on its amorphous form. Three different preparation in good agreement with that of the commercial material as
techniques, such as quench cooling of the melt, ball milling published recently by Ghugare et al.16 Indapamide is soluble in
and cryogrinding, were applied to convert the crystalline acetonitrile, ethyl acetate, glacial acetic acid, methanol, ethanol
indapamide into its amorphous form. To confirm the and other alcoholic solvents. It is very slightly soluble in ether
amorphous nature of the examined materials, X-ray powder and chloroform, while practically insoluble in water.
diffraction (XRD) and differential scanning calorimetry (DSC) Amorphous Samples Preparation. Quench Cooling of
were used. The DSC was also applied to determine the glass the Melt. Crystalline IND hemihydrate was placed on a
transition temperature (Tg) of amorphous IND. The results of stainless steel plate and heated on a hot plate until a complete
FT-IR experimental technique give us information about the melt was achieved (the sample becomes a yellow, transparent
chemical stability of indapamide drug. Furthermore, dielectric film). The molten sample was then quenched by being put onto
spectroscopy (BDS), which is often applied to study liquid− a cold tile.
glass transitions in various pharmaceuticals, was employed to Ball Milling. The room temperature ball milling was
characterize the molecular dynamics of the analyzed diuretic performed using a Planetary Ball Mill (Retsch, Germany). A
agent. Experimental results obtained were supplemented by zirconium jar was filled with the examined material and 6
atomic density functional theory (DFT) calculations. Finally, zirconia balls (20 mm in diameter). The rotation speed was set
the apparent solubility studies of amorphous IND samples were to 400 rpm. We have performed three separate tests with the
performed highlighting crystallization tendency of amorphous same amount of material (6 g) applying different grinding times.
phase in aqueous media. Each milling cycle lasted 15 min and was followed by a 5 min break.
3613 dx.doi.org/10.1021/mp400116q | Mol. Pharmaceutics 2013, 10, 3612−3627
Molecular Pharmaceutics
Total milling times of indapamide were 1 h, 2.5 h and 5 h. After
milling, indapamide becomes a yellow powder.
Cryogenic Grinding. Cryogenic grinding of indapamide was
carried out by means of a 6770 SPEX freezer/mill. The total
mass of the milled indapamide was 3 g. The sample was placed
in a stainless steel vessel and was immersed in liquid nitrogen.
The stainless steel rod present in the vessel is vibrated by
means of a magnetic coil. Prior to the start of grinding, the
sample was subjected to 10 min of precooling. The mill was set
to function at an impact frequency of 15 Hz. Five minute
grinding intervals were separated by three minute cool-down
periods. The effective grinding times were 15, 30, and 45 min.
After the cryogenic grinding, the vessel with the ground sample
was equilibrated in a vacuum oven at 25 °C, until room
temperature was reached. After milling, indapamide becomes a
yellow powder.
Analytical Techniques. XRD measurements. XRD experi-
ments were performed at ambient temperature on a Rigaku-
Denki D/MAX RAPID II-R diffractometer (Rigaku Corpo-
ration, Tokyo, Japan) equipped with a rotating anode Ag KR
tube (λ = 0.5608 Å), an incident beam (002) graphite
monochromator and an image plate in the Debye−Scherrer
geometry. The pixel size was 100 μm × 100 μm. Studied
samples were placed inside Lindemann glass capillaries (1.5 mm
in diameter). Then, the measurements were performed on
sample-filled and empty capillaries, and the background
intensity of the empty capillary was subtracted from the sample
signal. The beam width at the sample was 0.1 mm. The two-
dimensional diffraction patterns were converted into one-
dimensional intensity data using suitable software.
Nuclear Magnetic Resonance (NMR). The 1H and 13C
NMR spectra were recorded at 400 and 100 MHz, respectively,
on a Bruker 400 (Germany).
1
H NMR (400 MHz, DMSO-d6): δ = 1.30 ppm (d, J =
6.1 Hz, 3H, CH3); 2.59 (dd, J = 15.3 Hz, J = 11.1 Hz, 1H, CH2);
3.17 (dd, J = 15.3 Hz, J = 8.1 Hz, 1H, CH2); 3.93−3.99 (m, 1H,
CH); 6.50 (d, J = 7.8 Hz, 1H, CH); 6.75−6.78 (m, 1H, CH);
7.02−7.06 (m, 1H, CH); 7.11 (d, J = 7.2 Hz, 1H, CH); 7.74
(s, 2H, NH2); 7.80 (d, J = 8.3 Hz, 1H, CH); 8.12 (dd, J =
8.3 Hz, J = 2.2 Hz, 1H, CH); 8.51 (d, J = 2.2 Hz, 1H, CH);
10.53 (s, 1H, NH).
13
C NMR (100 MHz, DMSO-d6): δ = 18.5 ppm (CH3); 35.4
(CH2); 62.9 (CH); 108.7 (CH), 119.9 (CH); 124.4 (CH);
127.0 (CH); 127.1 (C); 128.3 (CH); 131.6 (C); 131.7 (CH);
132.1 (CH); 133.6 (C); 141.3 (C); 151.4 (C); 163.9 (CO)
Broadband Dielectric Spectroscopy (BDS). Ambient
pressure dielectric measurements of glassy and supercooled
indapamide were performed over a wide frequency range from
10−1 to 106 Hz using a Novo-Control GMBH Alpha dielectric
spectrometer. For the isobaric measurements, the sample was
placed between two stainless steel electrodes of the capacitor
(diameter 20 mm) with a gap of 0.1 mm. The dielectric spectra
were collected in a wide temperature range from 133 to 441 K.
The temperature was controlled by the Novo-Control Quattro
system, with the use of a nitrogen gas cryostat. Temperature
stability of the samples was better than 0.1 K.
Differential Scanning Calorimetry (DSC). Calorimetric
measurements of crystalline and amorphous indapamide were
carried out by Mettler-Toledo DSC apparatus equipped with a
liquid nitrogen cooling accessory and a HSS8 ceramic sensor
(heat flux sensor with 120 thermocouples). Temperature and
enthalpy calibrations were performed using indium and zinc
standards.
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Sealed aluminum crucibles (40 μL) with lids containing five
punctures to enable water to volatilize during heating were used
for all samples. Standard DSC measurements were performed at a
heating rate of 10 K/min under a nitrogen purge (60 mL/min).
Moreover, using a stochastic temperature-modulated differ-
ential scanning calorimetry (TMDSC) technique implemented
by Mettler-Toledo, TOPEM , the dynamic behavior of the
glass−liquid transition of the studied materials has been
analyzed in the frequency range from 4 mHz to 40 mHz in
one single measurement at a heating rate of 0.5 K/min. In the
experiment, the temperature amplitude of the pulses of 0.5 K
was selected. The calorimetric structural relaxation times τα =
1/2πf were determined from the temperature dependences of
the real part of the complex heat capacity cp′(T) obtained at
different frequencies in the glass transition region. The glass
transition temperature Tg was determined for each frequency as
the temperature of the half step height of Cp′(T).
Infrared Measurements (FT-IR). Infrared measurements
were performed using a Bio-Rad FTS-6000 spectrometer
equipped with a KBr beam splitter, a standard source and a
DTGS Peltier-cooled detector. The single crystalline and glassy
spectra of indapamide have been collected using GladiATR
diamond accessory (Pike Technologies) in the range from 400
to 4000 cm−1. The temperature measurements were carried out
using a heated plate of the GladiATR diamond accessory (Pike
Technologies) equipped with a temperature controller with
accuracy ±0.1 K. Time-dependent spectra were recorded every
10 min. All spectra were recorded with a spectral resolution of
2 cm−1. Presented results are an average of 16 scans in order to
ensure good quality spectra.
Mass Spectrometry. Liquid chromatography−mass spec-
trometry (LC−MS) was carried out using a Thermo Accela
Liquid chromatography coupled to a Thermo LTQ-XL-
Orbitrap Discovery mass spectrometer as the detector. The
column used for chromatographic separation was a Waters
Acquity HSS T3 C18, 2.1 mm × 150 mm 1.8 μm at operating
at 30 °C. A flow rate of 100 μL/min was used with an injection
volume of 4 μL. The mobile phase consisted of two solutions:
0.1% v/v formic acid in HPLC grade water as phase A and 0.1%
v/v formic acid in HPLC grade acetonitrile as phase B. The
total run time was 10 min, and the following gradient method
was used: 20% phase B to 80% phase B over 8.00 min, hold
until 7.00 min, then 20% phase B at 7.01 min and equilibrate
for 3 min. The LTQ-XL ion trap mass spectrometer was
coupled to the Accela LC system via an electrospray ionization
(ESI) probe. The capillary temperature was maintained at
310 °C, sheath gas flow rate 60 arbitrary units, auxiliary gas flow
rate 5 arbitrary units, sweep gas flow rate 0 arbitrary units,
source voltage 3.20 kV, source current 100 μA, capillary voltage
48 V and tube lens 82 V. Indapamide was detected in negative
ion mode, and its retention time was 6.6 min. Full FTMS
scanning (m/z 80−1000) was used to detect degradation
products. The samples as methanolic solutions were also
infused directly into the ion chamber at a flow rate of 5 μL/min
using a Hamilton syringe.
Apparent Solubility Measurements. HPLC Equipment
for the Solubility Assay. In this study, a high performance
liquid chromatography HPLC system (Shimazu LCsolution
Chromatography System, model Prominence LC-20A) was
used to quantify the amount of indapamide dissolved. The
system was equipped with a UV/vis detector (model SPD-20 A),
a quaternary gradient pump (model LC-20 AD) with a parallel-
type double plunger, a degasser (model DGU-20 A5) with a
dx.doi.org/10.1021/mp400116q | Mol. Pharmaceutics 2013, 10, 3612−3627
Molecular Pharmaceutics
fluoroethylene membrane, an autosampler (model Sil-20 A HT)
with two trays for standard 1.5 mL vials and a column oven
(model CTO-1-AS VP). A Microsorb 100-5 C8 (S150 × 4.6,
Varian USA) column was used. The column was thermostatted
at 313 K. The mobile phase was composed of water/methanol/
acetic acid (55:45:0.1 v/v) and used at flow rate of 1.0 mL/min.
20 μL sample volumes were injected into the column, and
elution was monitored at 240 nm. Shimadzu LC solution
software (ver. 1.25) data processing software was used to record
and integrate the chromatograms.
Apparent Solubility Study of IND. To determine the
solubility of crystalline and amorphous forms of indapamide
in purified water, phosphate buffer pH 6.8 and 0.1 M
hydrochloric acid, about 100 mg of each sample was added
to 50 mL of each medium. The resulting slurry was strirred for
24 h at 25 °C ± 0.5 °C (I series) and 37 °C ± 0.5 °C (II
series). The samples were filtered through a sterile syringe filter
(25 mm, PET-polyester, pore size 0.45 μm) from Macherey-
Nagel (Bioanalytic, Poland). Each filtrate was diluted 1:200 v/v
(0.25 mL of the filtrate solution was made up to 50 mL in a
volumetric flask). In order to prevent precipitation of
indapamide from the saturated solutions, all the flasks were
heated to 25 or 37 °C.
Apparent solubility of the various forms of indapamide was
determined by HPLC (n = 9) as described above. The
concentration of indapamide was calculated from a rectilinear
regression equation obtained from measurements of 10 standard
solutions in the concentration range of 0.025−2.0 μg/mL. The
calibration curve, y = 72615x − 355.8 (R2 = 0.9999), was
determined from the relationship between the peak area of the
chromatograms and the concentration of standard solutions. The
retention time of indapamide was 6.5 min.
Nonsink Dissolution Studies. Nonsink dissolution studies
were performed in deionized water at 37 °C. 50 mg of the
tested material was placed in a glass vial (diameter, 22 mm;
height, 70 mm) containing 25 mL of the medium and stirred
(Stuart SD162, U.K.) using a magnetic bar (diameter, 4 mm;
length, 12 mm) at a rate of 1300 rpm. The vial was placed in a
double walled jacketed beaker connected to a water bath
(Haake F3, Germany). Sampling of the liquid medium was
carried out using a circulation system composed of an LSMatec
peristaltic pump fitted with a 10 μm filter (HMWPE, Porex
Technologies, Germany). The filtered solution was passed
through a 2 mm flow-through UV quartz cuvette placed in a
Shimadzu UV-1700 PharmaSpec spectrophotometer (Japan)
set to 279 nm. The results are an average of three measure-
ments, and error bars present standard deviations. For statistical
comparisons, one-way analysis of variance (ANOVA) followed
by the post hoc Tukey’s test was used. Differences were
considered significant at p < 0.05.
■
CALCULATION METHODS
All quantum chemistry calculations for indapamide (IND)
molecule were performed with the use of density functional
theory in the ORCA package.17 In the first step we have
performed geometry optimizations of a dozen random IND
structures with the use of hybrid B3LYP functional and the
6-31g* basis set. Three the most stable structures were then
reoptimized using the same functional and 6-31++g(2d,2p)
basis set. The structure presented is in the minimum
determined by the position of the sulfonamide (−SO2NH2)
group, which is the most flexible part of the molecule. In order
to simulate the rotation at the B3LYP/6-31+g(2d,2p) level of
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theory, relaxed geometry scan by changing proper dihedral
angle was performed. Thereafter, structures characterized by
the highest value of electronic energy were optimized as
transition states at the same level by means of the eigenvector
following method. Transition states and minima were
subsequently confirmed by performing vibrational analysis.
Frequencies were calculated numerically at the same level of
theory. Molecules were visualized using Avogadro package.18
■ RESULTS AND DISCUSSION
Part A: Study of the Amorphous IND Obtained by the
Vitrification Method. Various methods for preparation of
pharmaceutical systems in their amorphous forms have been
reported, such as freeze-drying, spray drying or mechanical
milling, however the simplest approach is based on the rapid
cooling of the molten sample, typically referred as vitrification.
However, this method carries a risk associated with chemical
decomposition of the drug during melting of the crystalline
material. It should be emphasized that if thermal degradation
occurs, the decomposed amorphous material obtained is
disqualified as a therapeutic agent. This is the reason why
chemical purity of drugs subjected to thermal stress conditions
in a manufacturing process should be addressed with particular
care.
Chemical Stability Analysis. To determine the melting
point of IND, differential scanning calorimetry was applied.
The DSC curve obtained during heating of the crystalline
compound up to 473 K is depicted in Figure 2. An endothermic
peak, with an onset at 437 K, indicates melting of the sample.
The melting point (Tm) value of IND obtained in this study is
in good agreement with that determined recently by Ghugare
et al.16 Since the commercial form of the examined compound
contains 2.5% water, in the thermogram presented in Figure 2
one can also observe a broad endotherm related to evaporation
of water. To produce an amorphous form of the drug,
indapamide was molten and then cooled to room temperature.
The subsequent heating from the glassy state shows the
characteristic signature for the glass transition in the temper-
ature dependence of the heat flow at 377 K. Moreover, there
was no thermal effect associated with evaporation of water,
indicating that indapamide obtained by vitrification is
anhydrous. To confirm the disordered nature of the amorphous
form of IND, the XRD technique, being one of the most
definitive methods for detecting and quantifying molecular
order in a system, was applied. As illustrated in Figure 3, the
very broad halo presented as a black line, compared to the
sharp peaks typical of the crystalline state (blue line), indicates
that the vitrified sample is indeed an amorphous material.
It should be noted that the amorphous indapamide obtained
by means of vitrification becomes yellow in contrast to the
white crystalline form. Such observation might indicate that the
examined drug undergoes thermal decomposition upon heating.
To verify this supposition FT-IR was employed, which is able to
detect chemical changes occurring in a sample. First, we have
performed the analysis of crystalline IND as a reference. As
illustrated in Figure 4, in the FT-IR spectrum of the crystalline
compound one can distinguish several characteristic regions.
The first one is located between 3700 and 3300 cm−1. There are
two sharp absorption bands associated with the symmetric
(3499 cm−1) and asymmetric (3655 cm−1) stretching vibrations
for the hydroxy functional group of water molecules observed
in the commercial IND sample. The sulfonamide group has
strong bands due to its NH2 stretching vibrations at 3431 and
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Molecular Pharmaceutics
Figure 3. X-ray diffraction patterns for various solid-state forms of
IND performed at room temperature T = 298 K.
Figure 4. FT-IR spectra collected during annealing of IND from 293
to 453 K. As a reference the spectrum of the starting, crystalline
material recorded at room temperature is presented as a bold red line.
Dashed lines indicate bands undergoing changes during annealing.
The upper panel presents FT-IR spectra of IND heated to 16 K above
the melting point for one and two hours, respectively.
3342 cm−1 in the same infrared region. Moreover, a peak at
3312 cm−1 of NH associated with the amide moiety of the
indapamide molecule is observed. The second characteristic
region in the FT-IR spectrum is observed between 1700 and
1000 cm−1. One of the strongest bands visible in this range is
located at 1655 cm−1 and arises from the carbonyl stretching
vibration. Other band assignments with their wavenumber
positions for crystalline IND phase are summarized in Table 1.
It should also be noted that infrared spectroscopy has recently
been applied by Ghugare et al. to analyze the crystalline form of
indapamide.16 Despite the fact that the authors16 dispersed
analyzed diuretic drug in KBr, the published diffuse reflectance
infrared Fourier transform spectrum of IND is in good agree-
ment with our results.
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In the next step, FT-IR spectra were recorded in a wide
temperature range, from 293 up to 453 K. As one can see in
Figure 4, with increasing temperature, broadening of bands and
the loss of spectral resolution are observed. Both of these effects
are typical for the melting process. Furthermore, there are a few
distinct differences in FT-IR spectra between the crystalline and
molten samples. The most significant changes are observed in
the 3700−3200 cm−1 region where two well resolved bands
assigned to vibrations of water molecules are detected i.e. at
3655 and 3499 cm−1. As illustrated in Figure 4, their intensity
decreases with increasing temperature, and finally, close to the
melting point, the bands are undetectable. It means that the
amount of water in the sample decreases with increasing
temperature. To better illustrate this process, the integral intensity
of two water bands was calculated and plotted versus temperature
(Figure 5). From Figure 5 it can be clearly seen that in fact the
whole water evaporates below 373 K. This result is in good
agreement with calorimetric measurements presented previously.
The other noticeable change in the temperature dependent IR
spectra, presented in Figure 4, is associated with the N−H band
at 3312 cm−1 of the crystalline sample. As it can be seen, with
increasing temperature the band becomes less intense and
disappears at 453 K. Simultaneously with rearrangement of the
N−H environment, the stretching band of the carbonyl moiety
shifts toward higher wavenumbers and a new band at 1458 cm−1 is
found to appear. According to the theoretical calculation of IND
spectrum, this new band is attributed to the bending C−OH
vibrations. All these changes indicate that the increase of
temperature induces the proton transfer from the nitrogen to
the oxygen atom of the carbonyl group. Consequently, the double
bond formerly placed between carbon and oxygen atoms shifts to
the carbon−nitrogen position (νCN at 1662 cm−1). It means
that indapamide molecule may exist in equilibrium with its two
easily interconvertible constitutional isomers (see Figure 1).
Taking into account the types of bonds involved in this proton
transfer, the transition observed in IND may be called an amide−
imidic acid tautomerism.
Since the FT-IR spectra recorded showed only the
tautomeric transformation and did not reveal any decom-
position at Tm, in the next step of our experiments we have
annealed the examined sample at 453 K i.e. 12 K above its
melting point. The spectra collected after one and two hours of
heating are presented in Figure 4. It is clearly seen that there is
no difference between the isothermally annealed samples.
Taking into account the previous IR results one can expect that
the indapamide is a thermally stable compound. The thermal
decomposition of the examined material was also excluded by
HPLC−MS and NMR measurements performed in this study
(a detailed NMR description of this sample is presented in the
Experimental Section). The above findings are additionally
supported by the thermogravimetric analysis (TGA) presented
in a previous study.16 The TGA pattern indicates that the onset
of thermal decomposition of IND, related to the onset of the
mass loss of IND, begins just above 560 K, well above the
melting point temperature. Consequently, one can conclude
that it is possible to prepare the pure amorphous IND by
means of vitrification. At the same time the change in color of
IND drug during melting cannot be associated with chemical
decomposition of the sample. It is worth noting that dissolution
of indapamide in DMSO or ethanol at room temperature also
results in the change of solution color from colorless to yellow.
Thus, the peculiar behavior of IND changing color may be an
effect of IND transition from crystalline to the liquid phase. It is
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Molecular Pharmaceutics Article

Table 1. FT-IR Band Assignments with Their Wavenumber Positions for Crystalline and Glassy IND Samplesa
crystalline IND amorphous IND
vibration wavenumber [cm−1] vibration wavenumber [cm−1]
νasym H2O 3655 νOH 3604
νsym H2O 3499
νasym NH2 3431 νasymNH2 3387
νsym NH2 3342 νsymNH2 3277
νNH 3312
νCN; νOH 1662
νCO; νNH 1655
νasymCC(benzene); νCH 1611 νasymCC(benzene); νCH 1607
δNH2 1563 δNH2 1561
βCH; βNH 1460 βCH; βCOH 1458
βCH3; γNH2; νasym(SO)2; βCH 1323
νasymCC at Cl; γNH2; νasym(SO)2 1297 γNH2; νasym(SO)2; βCH 1295
νNN; βNH; βCH 1281
βNH; βCH 1242 βCOH; βCH; βCH2 1242
γNH2; νsymCCN(pyrrole ring) 1067 γCH2; γNH2; γCH3; γNH2; νsym(SO)2 1090
ωNH2; νNS 847 ωNH2; νNS; βCOH; νNC 841
ωNH2; νNS; βCOH; νCH 800
a
δ, twisting; ω, wagging; γ, rocking; β, bending; ν, stretching; asym, asymmetric; sym, symmetric vibrations.

well as theoretical studies of the tautomerization reaction.21−25

Figure 5. The temperature dependence of integral intensity for two
bands, at 3655 and 3499 cm−1, respectively, recorded during heating of
crystalline IND from room temperature to 453 K.
noteworthy that there are several compounds that change their
color after melting or dissolution. As an example one can
mention indomethacin19 or piroxicam,20 which like indapamide
become yellow after amorphization. However, it should be
emphasized that in the latter case the tautomerization reaction
was found to be responsible for changing the color of sample.
Since indapamide also reveals the isomerization tendency, one
cannot exclude that the yellow color of the sample is due to the
tautomerisation, which occurs during melting.
To provide additional information about the tautomerization
reaction of amorphous IND, FT-IR spectroscopy was employed
once again. The infrared spectrum of vitrified indapamide
sample is depicted as a top green line in Figure 6. Since this
spectrum practically overlaps that which was recorded for the
molten sample i.e. there are bands corresponding to νCN
and βC−OH vibrations, while the NH band associated with the
amide moiety of indapamide molecule is almost absent, one can
conclude that the tautomeric equilibrium achieved at the
melting temperature is conveyed into the amorphous material. In
recent years, there has been a growing interest in experimental as
3617
This is due to likely serious consequences of the proton transfer.
Especially, isomerization of pharmaceuticals becomes an
important problem as various tautomers of one drug may reveal
different pharmacological activity. A frequently used antibiotic,
erythromycin, exhibits as many as three isomers,26 however only
one of them, the ketone form, reveals the antibacterial activity.
Moreover, it cannot be excluded that tautomers with no desired
therapeutic activity may cause a number of side effects.
Therefore, the following question arises: Is the activity of the
imidic acid form of IND the same as that of the amide isomer?
Unfortunately, to answer this question thorough bioactivity
studies are required. However, one cannot exclude that the weak
diuretic activity of indapamide drug is due to the tautomeric
forms that appear after dissolving the tablet in the gastrointestinal
tract.
Analysis of Molecular Dynamics in Supercooled and
Glassy State of IND. In the literature one can find many
examples that demonstrate that studies of the molecular
mobility in amorphous pharmaceuticals help to control the
behavior of these systems at the macroscopic scale.27−30 The
most frequently used experimental methods for monitoring the
molecular dynamics of amorphous medicines are dielectric
spectroscopy, depolarized light scattering, dynamic mechanical
analysis, proton correlation spectroscopy or nuclear magnetic
resonance. Here, to provide the essential information about
the molecular mobility in the supercooled and glassy states of
indapamide, broadband dielectric spectroscopy (BDS) was
applied. Additionally, temperature-modulated differential scan-
ning calorimetry (TMDSC) was implemented to analyze the
dynamic behavior of IND as well as to determine its glass
transition temperature.
The imaginary part of complex dielectric permittivity spectra
measured at ambient pressure over seven decades of frequency
in the supercooled liquid state and the amorphous phase of
IND are shown in Figure 7. Panel a of this plot presents
dielectric loss spectra recorded above Tg: at temperatures from
385 to 441 K with a uniform increment of 4 K. In this
temperature range two dielectric processes can be distinguished:
(i) conductivity relaxation at low frequencies which is related to
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Molecular Pharmaceutics
Figure 6. FT-IR spectra of IND: crystalline, obtained by quench-cooling, obtained by cryogenic grinding and prepared by room temperature milling.
All spectra were collected at room temperature.
Figure 7. Dielectric loss spectra of IND obtained by quench cooling of
the melt. Panel a presents dielectric loss above the glass transition
temperature, whereas in panel b dielectric loss spectra collected below
Tg are presented.
the translational motion of ions and (ii) the structural relaxation
process related to the cooperative rearrangements of indapamide
molecules. In contrast to other pharmaceuticals, like glibencla-
mide or indomethacin, the dc contribution for indapamide is not
significant, resulting in the maximum of structural relaxation peak
being well resolved despite close proximity to the glass transition
region. As illustrated in the upper panel of Figure 7 the shift of
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the α-relaxation peak toward lower frequencies is related to a
decrease of temperature. This behavior reflects the slowing down
of cooperative molecular motions of IND. Below Tg the
structural relaxation moves out of the frequency measurement
window, and then a secondary relaxation, called the γ-process,
becomes apparent (see Figure 7b). Similar to the mentioned
α-relaxation, the γ-mode also slows down during cooling, but it is
far less sensitive to the change of temperature. To move it over
6 decades in frequency, it was necessary to decrease the
temperature by 140 K, from 273 to 133 K. The nature of this
secondary mode will be discussed latter.
One of the most important parts of the dielectric spectra
analysis is to investigate the shape of the structural relaxation
process. From the pharmaceutical point of view, this parameter
is especially important as it was found to be directly related to
the physical stability of the amorphous material. As suggested
by Shamblin et al.,31 the stability of various amorphous drugs
should increase as the α-peak becomes narrower, i.e. with
increasing βKWW parameter. Examples of active substances
obeying this rule are e.g. nonivamide,32 telmisartan33 or
glibenclamide.34 To verify whether this relationship is valid
also in the case of indapamide, we first constructed a so-called
“masterplot” in which a number of dielectric curves measured at
different temperatures (both above and below Tg) have been
superimposed. As the reference one we have used the spectrum
recorded at T = 385 K. It can be clearly seen in Figure 8 that
the shape of the α-process is practically invariant upon cooling.
Thus, in the case of IND, similarly to many other glass-forming
liquids, the time−temperature superposition principle is
fulfilled. Additionally, it can be seen that the α-relaxation
process of IND is asymmetric and broader than the classical
Debye response. To parametrize the shape of this mode we
have applied the Kohlrausch−Williams−Watts (KWW)
function:35,36
⎡ ⎛ ⎞ βKWW ⎤
t
ϕ(t ) = exp⎢ −⎜ ⎟ ⎥
⎢⎣ ⎝ τα ⎠ ⎥⎦
(1)
where t is the time, τσ is the characteristic structural relaxation
time and βKWW (0 < βKWW ≤ 1) denotes the stretching
parameter, which is related to the width of the relaxation peak.
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Molecular Pharmaceutics
Figure 8. Superimposed dielectric spectra of IND taken at ambient
pressure (p = 0.1 MPa), at seven different temperatures above and
below Tg. The solid red line is the KWW function with βKWW = 0.72,
while the blue line is a Cole−Davidson fit (α = 1, β = 0.63). The green
arrow denotes the position of the primitive relaxation calculated from
eq 3 at 385 K. The inset panel presents the spectra collected at 363 K
during physical aging of IND .
Since the above equation refers to the time not the frequency
domain, to describe the ε″( f) peaks the one-sided Fourier
transform of the KWW function has to be applied:
∞⎛ dΦ ⎟⎞
ε″(ϖ) = Δε ∫0 ⎜
⎝
−
dt ⎠
sin(ϖt ) dt
(2)
where Δε is the dielectric strength of the α-relaxation. The
representative fit of the KWW function to the experimental
data, with the βKWW exponent equal to 0.72, is shown as a red line
in Figure 8. Since the obtained value of βKWW is quite high, one
can suppose that amorphous indapamide should not crystallize.
This assumption is supported by experimental data because
neither dielectric nor calorimetric measurements reveal crystal-
lization signs during heating of the amorphous IND sample.
From the dielectric spectra analysis presented in Figure 8 it is
also evident that the KWW function describes the data well
only in the vicinity of the α-peak maximum. It can be easily
seen that at frequencies about two decades higher than the
maximum of the α-process the experimental data systematically
deviate from the KWW fit. The origin of such a trend, usually
called “excess wing”, has remained a matter of hot debate in the
past decade.37−41 Some scientists interpreted this deviation as
an inherent part of the α-relaxation42 while others said that the
excess wing is a high frequency flank of the secondary relaxation
process hidden under the dominant α-peak.43,44 According to
the second explanation, for the first time postulated by Ngai45
and experimentally verified by Lunkenheimer46 and other
researchers, this unresolved secondary mode originates from
some local motions of the entire molecule, and it is believed to
be a precursor of α-relaxation. To determine the maximum of
this process, usually called the Johari−Goldstein relaxation (JG),
the coupling model (CM) approach is generally applied,47
τ0 = (tc)1 − βKWW (τα)βKWW (3)
where τ0, usually called the primitive relaxation time, denotes the
possible relaxation time of the hidden secondary mode while the
parameters τα and βKWW characterize the α-relaxation, and tc = 2 ps.
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This procedure has been applied several times in the past to
predict the maximum of the hidden β-process. Using the values
of βKWW = 0.72 and the relaxation time determined from the
maximum of the α-peak recorded at 385 K, we have calculated
τ0 for indapamide. The τ0 was found to be equal to 2.2 × 10−4 s,
which corresponds to the frequency f 0 = 714 Hz. As illustrated in
Figure 8, the value of f 0 determined lies within the frequency
range where the excess wing is observed (green arrow).
In order to provide more information about the nature of
this excess wing visible in dielectric spectra of indapamide, we
have also performed an aging experiment. It is well-known that,
except for the high pressure measurements,48,49 this is one of
the most frequently used methods to explore more thoroughly
the origin of excess wing.50 If upon physical aging the excess
wing transforms into a well-separated peak (or shoulder), this
provides evidence that the excess wing may be classified as a
JG-type relaxation. During the aging experiment the examined
diuretic sample was annealed isothermally at 363 K, close to Tg.
Both the first and the last dielectric spectra (recorded at the
beginning and after 22 h of annealing) are depicted in the inset
panel of Figure 8. As one can see, the excess wing becomes more
pronounced with time. This experimental result suggests that the
observed process may be attributed to the JG-relaxation.
The dielectric α-relaxation peak of IND can be also analyzed
by means of the Havriliak−Negami function with the dc-
conductivity term added that fits the experimental data more
precisely than the KWW function.51
σdc Δε
ε″ (ϖ ) = + ε∞ +
ε0 ϖ [1 + (iωτHN)αHN ]βHN (4)
where ε∞ is the limiting high-frequency permittivity, τHN
denotes a characteristic relaxation time; exponents αHN and
βHN characterize symmetric and asymmetric broadenings of
the dielectric loss curve; Δε is relaxation strength and ω is an
angular frequency. A representative fit of eq 4 to the dielectric loss
curve recorded above Tg is depicted as a blue line in Figure 8. To
parametrize the dielectric loss spectra recorded above Tg, the HN
function with αHN =1, which is usually called the Cole−Davidson
(CD) function,52 was applied. As one can see, the CD equation
describes the α-peak better than the previously used KWW
function. However, to obtain the perfect fit in whole measured
range, the effect of the unresolved β-mode should be taken into
account. Consequently, the superposition of the Cole−Davidson
and Cole−Cole function has to be applied. Herein, it should be
noted that the Cole−Cole function, i.e. when the parameter βHN is
set to one, describes well symmetric and broad relaxation modes.
Thus, it can also be successfully used to characterize the secondary
γ-relaxation process of IND.
In the next step of the dielectric data analysis, based on the
CC and CD fit parameters determined previously, the character-
istic relaxation times of both modes, α and γ, observed in
permittivity spectra of IND were calculated according to eq 5:53
⎡ ⎛ α ·π ⎞⎤−1/ αHN ⎡ ⎛ α ·β ·π ⎞⎤1/ αHN
τ = τHN⎢sin⎜⎜ HN ⎟⎟⎥ ⎢sin⎜⎜ HN HN ⎟⎟⎥
⎢⎣ ⎝ 2 + 2βHN ⎠⎥⎦ ⎢⎣ ⎝ 2 + 2βHN ⎠⎥⎦
(5)
The experimentally determined variations in log τα and log τγ with
temperature are depicted as solid squares and circles, respectively,
in the form of a so-called Arrhenius plot shown in Figure 9.
As illustrated in this figure the log τα(1/T) dependence can be
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Molecular Pharmaceutics
Figure 9. Relaxations map of vitrified IND. Temperature dependence
of structural and secondary γ-relaxation relaxation times are depicted
as solid squares and circles, respectively. Solid lines are VFT and
Arrhenius fits to the experimental data. Fitting parameters are
presented in Table 2. Additionally, solid triangles are the structural
relaxation times determined from TMDSC measurements for
anhydrous IND.
reasonably well described over the entire measured range by
means of the Vogel−Fulcher−Tammann equation,54−56
⎛ DT0 ⎞
τα = τ0 exp⎜ ⎟
⎝ T − T0 ⎠ (6)
From the fit of the VFT equation to τα(T) dependence one can
easy determine the glass transition temperature of IND. Applying
the most frequently used definition, which describes Tg as the
temperature at which the dielectric relaxation time τα reaches
100 s, the glass transition temperature of IND was found to be
equal to 374 K, and this value well agrees with the one found from
DSC measurements (TgDSC = 377 K). Additionally, in this paper
we have determined the structural relaxation times of indapamide
from the temperature dependences of the real part of the complex
heat capacity Cp′ measured by the stochastic temperature
modulated DSC technique. As illustrated in Figure 9, DSC data
are in good agreement with the structural relaxation times
determined from the dielectric measurements (Table 2).
The most characteristic feature of molecular dynamics in
vitrifying liquids is non-Arrhenius dependence of the τα(T)
curve. However, the degree of deviation from Arrhenius
behavior varies from one compound to another. To assess
whether or not this divergence is significant, the “steepness
index” or “fragility” defined by Bohmer et al.,57
d log τα
m≡
d(Tg /T )
T = Tg (7)
is usually calculated. Using this parameter we can classify
supercooled liquids into two types: (i) “strong”, if the
temperature dependence of structural relaxation times is close
to Arrhenius behavior (m ≤ 30), and (ii) “fragile”, if log τα(1/T)
deviates significantly from the straight line (m ≥ 100). When m
falls within the 30 < m < 100 range, the liquid is classified as
intermediate glass-former. Fragility calculations performed for IND
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Table 2. The Parameters of Fitting to the VFT and
Arrhenius Equations for Anhydrous and Hydrated Lidocaine
HCl Samples
sample vitrified IND cryomilled IND ball-milled IND
α-Process VFT Parameters
Tg (τ = 100 s) [K] 374.6
mP (τ = 100 s) 76 ± 2
log τ0 −16.9 ± 0.2
D 13.6 ± 0.6
T0 [K] 285 ± 2
υ-Process Arrhenius Dependence
log τ∞−υ −15.05 ± 0.06 −15.45 ± 0.13
Ea−υ [kJ/mol] 52.4 ± 0.3 54 ± 0.6
γ-Process Arrhenius Dependence
log τ∞−γ −13.33 ± 0.5
Ea−γ [kJ/mol] 32.9 ± 0.2
give m = 76. This value is close to those found for other recently
investigated amorphous pharmaceuticals such as verapamil
hydrochloride (m = 88),10 telmisartan (m = 87),33 glibenclamide
(m = 78)34 or indomethacin (m = 83).19 Thus, indapamide
investigated in this work can be classified as an intermediate glass-
former drug.
Now we consider the molecular mobility in the glassy state of
anhydrous indapamide that is reflected in secondary relaxation
processes. As one can see in Figure 7, except for the excess
wing, identified previously as a hidden β-process, in the
dielectric spectra of IND only one secondary relaxation (γ) can
be found. Since log τγ is linearly related to the inverse of
temperature, it is possible to fit the Arrhenius equation (eq 8)
to the experimental data and determine the activation energy
barrier of this mode.
⎛E ⎞
τγ = τ∞ exp⎜ a ⎟
⎝ RT ⎠ (8)
The activation energy barrier was found to be equal to
34 kJ/mol. This quite low value of Ea, together with the
symmetrical shape of the γ-peak, suggests that the motions of a
small part of indapamide molecule are responsible for this
secondary relaxation. In order to confirm this supposition we
have performed the DFT calculations. Since the sulfonamide
group seems to be the most interactive part of the indapamide
molecule, we have studied the conformational changes of this
moiety. Initially we have checked whether the dipole moment is
changing during the conversion or not. It should be emphasized
that the variation of the dipole moment is necessary to observe
intramolecular motions as a secondary mode in the dielectric
spectrum. As illustrated in Figure 10, the rotation of the
sulfonamide group of indapamide causes a change in the dipole
moment. That is why this movement may be considered as an
origin of the γ-relaxation. The value of activation energy of the
sulfonamide group rotation obtained in the B3LYP/6-31+
+g(2d,2p) model is equal to 25 kJ/mol, and it is slightly lower
than the value of Ea determined experimentally.
Theoretical Prediction and Experimental Verification
of the Physical Stability of Quenched Indapamide.
Understanding of the factors affecting crystallization from the
glassy state not only is important from a scientific perspective but
also has many practical applications e.g. in the pharmaceutical
industry. However, in contrast to the chemical stability
determination, where the standard analytical techniques are usually
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Molecular Pharmaceutics
Figure 10. Diagram representing changes of the dipole moment and
energy during rotation of the sulfonamide group of IND molecule.
applied, there is no certain methodology to predict the physical
stability of an amorphous material. Until now crystallization of the
glassy sample has been many times correlated to the degree of its
molecular mobility, thermodynamic properties (such as heat of
fusion, heat capacity, configurational entropy) or the methods of
amorphization. One of the parameters, considered as a key factor
affecting stability of an amorphous system, is dynamic fragility.
This is because the steepness index is related to an average degree
of molecular mobility reflected in a structural relaxation near the
glass transition. According to the TOP (two order parameter)
model proposed by Tanaka,58 near the glass transition region
competition between long-range density, being a driving force
toward formation of the crystals, and short-range ordering favoring
formation of the locally preferred structures is postulated. If the
second effect dominates, the system becomes “frustrated”.
Consequently, the crystallization is expected to be inhibited. It
has been shown that the degree of frustration can be related to
fragility, i.e. the lower frustrationthe greater fragilitythe
greater tendency to crystallize. Taking into account this
assumption, IND with the fragility index equal to 76 should be
classified rather as a system with high crystallization tendency. On
the other hand, as it was stated previously, we can distinguish only
one, well-pronounced secondary relaxation process of IND. It
means that the molecular mobility of the examined drug is
significantly retarded below the liquid−glass transition, which is
observed at a relatively high temperature (377 K). Consequently,
at room temperature conditions, i.e. almost 80 K below the Tg,
motions of IND molecules are deeply frozen, which prevents
crystallization of IND. Moreover, there is a general rule that says
that the long-term stability of amorphous drugs can be obtained by
storage at a temperature where molecular mobility associated with
the structural relaxation time approaches zero, i.e. at T0 in the
Vogel−Fulcher−Tamman (VFT) equation. This temperature was
found to be close to the so-called Kauzmann temperature,59 where
entropy of the supercooled liquid and crystal would be equal. In
the case of IND T0 is equal to 285 K. Thus, storage of IND at
such temperature conditions should guarantee its physical stability
for a typical shelf life.
To better understand the physical stability of the examined
amorphous diuretic agent we have to consider other factors
that may affect its crystallization. Specifically, the possibility
of isomerization should be also taken into account. As it was
mentioned in the previous part of this paper there are two
isomers coexisting in the glassy state of IND. Consequently, the
amorphous sample becomes a binary mixture in which less
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stable imidic acid forms are dispersed in the amide matrix. In
the literature one can find some reports that show that this kind
of equilibrium detected in the glassy state may strongly reduce
the tendency of such a system to crystallize. For example,
glibenclamide60 is a hypoglycemic agent with tautomerization
tendency or binary mixtures containing active pharmaceutical
substance and various excipients like polymers (PVP, PEG or
HPMC)61−63 or sugars (octaacetylmaltose)64 which are
characterized by highly physical stability in the amorphous
state. The above results indicate that factors influencing the
stability of an amorphous system should not be considered
separately, because only when studied together can they
provide full information about the physical stability of glassy
pharmaceuticals. Thus, in the context of molecular dynamics
investigations as well as the proton transfer in IND one can
suppose that the IND system examined should not reveal the
crystallization propensity from the amorphous state. To verify
these theoretical predictions, X-ray powder diffraction was used.
XRD measurements performed after one year of storage of the
sample at room temperature showed no sign of crystallization.
Part B: Study of Amorphous Indapamide Samples
Obtained by Cryogenic Grinding and Room Temper-
ature Milling. Mechanical milling of crystalline solids is a very
popular technique applied in many fields of industry to reduce
the particle size. It is also used in the pharmaceutical industry since
micronization was proven to enhance the dissolution properties
of drugs through an increase in the surface area of micro- or
nanoparticulates. As shown in the literature, the mechanical
treatment may also lead to amorphization of crystalline materials.
For instance, amorphization by milling was observed for
piroxicam,20 glibenclamide,34 sucrose or ziprasidone.65
Physicochemical Characterization of Milled IND
Samples. Herein, to produce the amorphous form of IND
drug we have applied two types of grinding: cryomilling, being
operated at very low temperatures using cryogenic media such
as liquid nitrogen, and ball-milling that is carried out at room
temperature. In the first case the IND sample (3 g) was milled
for 15, 30, and 45 min. On the other hand, the ball-milling
lasted for 1, 2.5, and 5 h. However, contrary to cryogrinding the
amount of milled sample was two times higher (6 g). The
diffraction patterns of all micronized materials are presented in
Figure 3. Significant broadening and decrease of intensity of
diffraction peaks attributed to reduction of sample crystallinity
was observed with increasing milling time. After 15 min of
cryogrinding and 1 h of ball-milling the degree of IND
crystallinity, calculated as a ratio of X-ray diffraction peak areas
for the partially crystalline sample to that being 100%
crystalline, was found to be equal to 34% and 36%, respectively.
However, when the milling time was extended to 30 min and
2.5 h, respectively, indapamide became almost completely
amorphous. The degrees of crystallinity were calculated as 5%
(cryomilled IND) and 8% (ball-milled IND). A complete
crystal−glass conversion was observed after 45 min of
cryomilling while at room temperature 5 h of grinding was
required to obtain fully amorphous IND. As illustrated in
Figure 3, Bragg peaks of these samples, characteristic for
materials with a long-range three-dimensional molecular order,
were replaced by broad halos identical to that recorded for
molten-quenched indapamide. Interestingly, amorphous IND
powders obtained using the mechanical treatment were yellow,
just like the quenched material.
The experimental data presented clearly indicate that cryomilling
is much more efficient in preparation of the glassy IND than
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Molecular Pharmaceutics
grinding at room temperature. The time needed to fully amorphize
crystalline IND by cryomilling was approximately seven times
shorter compared to with traditional room temperature milling.
This result agrees with the general rule that a decrease of the milling
temperature brings similar effects as an increase in milling intensity.
Additionally, it is worth pointing out that mechanical amorphization
of crystalline IND is much more efficient than milling of other
organic compounds reported in the literature. For example, full
amorphization of indomethacin19 and furosemide66 was achieved
after 60 and 120 min of cryomilling, respectively, while in the case
of IND only 45 min was necessary. Moreover, it should be
stressed that in our experiments the amount of milled material was
three times higher than those for mentioned pharmaceutics. On
the other hand, the mechanical treatment of IND at room temp-
erature brings the complete amorphization only after 300 min,
while the amorphous state of indomethacin and trehalose was
achieved after more than 1200 and 1380 min of traditional ball
grinding. Hence, it can be stated that the mechanical treatment of
IND at both room and liquid nitrogen temperatures works very
effectively. Thus, these methods can be successfully applied in the
pharmaceutical industry to produce IND in the amorphous form.
In the next step we have performed the DSC analysis of
milled indapamide samples. In Figure 2 DSC heating scans of
cryomilled and ball-milled IND are illustrated. In both cases, two
thermal events were observed within the examined temperature
range: the first one associated with water evaporation and the
second one ascribed to the glass transition. As seen in the inset
panel of Figure 2, a heat flow jump characteristic for a glass
transition detected in the thermograms of milled IND occurs
exactly at the same temperature as that of the quenched liquid, i.e.
377 K. However, in contrast to the anhydrous vitrified sample, the
cryomilled and ball-milled materials contain 1.3% and 2.5% water,
respectively. The reduced water content in cryomilled IND is due
to the fact that before grinding indapamide hemihydrate has been
dried at 363 K for 12 h. On the other hand, amorphous sample
obtained by means of room temperature milling contains the same
amount of water as the starting hemihydrate. Nevertheless, in both
cases the whole amount of water evaporates before the glass
transition temperature is achieved. It is interesting that in the DSC
thermograms of the tested samples there is no exothermic peak
ascribed to so-called “cold crystallization”, which is frequently
observed during the slow heating of milled materials. This result
suggests high stability of ground IND against crystallization.
Indeed, X-ray diffraction measurements performed one year after
sample preparation did not reveal any Bragg reflections. It means
that there is no difference in terms of the physical stability between
amorphous IND samples prepared by milling and vitrification
techniques. This result is interesting in the context of recent
reports of tri-o-methyl-β-cyclodextrin,66 nucleosides67 or gliben-
clamide34 which demonstrate the opposite behavior. Thus, one
can formulate the following question: What factors af fect the
stability of milled IND? As shown in part A of this paper, in the
case of the vitrified sample the proton transfer was found to be
one of the key factors determining the stability of quenched IND.
Accordingly, a further important question to pose is: Does the
milling induce the amide−imidic acid transformation of
indapamide or not? As shown in the literature, the high level of
mechanical energy used during milling may cause significant
changes in the structure of the ground material. These may be
observed especially when the sample is milled above the liquid to
glass transition temperature. Such a scenario was shown for a
number of pharmaceuticals such as chloramphenicol, cimetidine or
indomethacin, where polymorphic transformations were induced
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by ball-milling. On the other hand, cryomilling was believed not to
induce chemical changes in the milled material because liquid
nitrogen prevents local overheating of the sample. However,
Adrjanowicz et al.68 recently have shown that even cryomilling is
able to modify the chemical structure of ground materials.
Therefore, one can suppose that both room temperature milling
and cryogrinding can induce changes in the structure of
indapamide. To characterize the structure of cryomilled and ball-
milled materials in detail, FT-IR spectroscopy was exploited. The
FT-IR spectra of milled IND are presented in Figure 6 together
with the data obtained for the crystalline and vitrified samples. It is
clearly visible that the data pertaining to all amorphous materials
are almost the same. The only difference in the high wavenumber
region 3700−3200 cm−1 was the vibrations of water molecules
detected for the milled IND. This is because the milled samples,
in contrast to the quenched IND, contain water. On the other
hand, similarly to the quenched indapamide, significant loss in
intensity of the N−H stretching vibration at 3312 cm−1 was found.
Furthermore, new bands attributed to CN and C−OH
vibrations also appear in the spectra. These changes in the FT-IR
spectra indicate that milling of IND both at room temperature and
in cryogenic conditions leads to the conversion of the amide form
of drug to its imidic acid isomer.
There are contradictory literature reports on the tautome-
rization reaction of milled materials. On the one hand, there are
several examples when the less stable isomer appears after
grinding, like in the case of glibenclamide or piroxicam. In the
former case tautomerism is observed independently of the
grinding conditions while for the second compound the
transformation was detected after milling at the liquid nitrogen
temperature. On the other hand, these findings are in contrast
with the experiments performed with sugars. Descamps et al.
have shown that mutarotation of saccharides, being a ring−
chain tautomerization, does not occur in amorphous trehalose
or lactose samples obtained by milling at liquid nitrogen
temperature.68 It means that after grinding there is only one,
initial anomeric form. However, it should be stressed that in the
case of anhydrous lactose 96% of milled material was still in the
α-anomeric form. Thus, one can suppose that after milling 4%
of the investigated sample was converted into the β-form.
Molecular Mobility of Milled IND Samples. In Figure 11
we present the imaginary parts of dielectric permittivity spectra
plotted as a function of frequency during heating of cryomilled
and ball-milled IND from 153 K up to 443 K. To show the
whole sets of data more clearly, dielectric spectra of each
sample were divided into two panels presenting the relaxation
dynamics above and below the glass transition temperature. As
can be seen in panels c and d of Figure 11, the imaginary part of
the complex dielectric permittivity, recorded for both milled
materials, reveals a well-resolved secondary relaxation process,
called υ-peak, which was not observed in dielectric spectra
of the melt-quenched sample. Similar to the γ-relaxation, the
υ-process shifts toward higher frequencies on heating, but in
contrast to the γ-mode it is more sensitive to temperature
changes. This behavior suggests that the activation energy of
this υ-process is greater than that for the γ-relaxation. The close
inspection of the dielectric loss spectra collected at the same
temperature conditions (T = 216 K), for milled and quench-
cooled samples, shows that the amplitude of the new dielectric
mode is higher than that of the γ-relaxation (see Figure 12).
Moreover, both processes, γ and υ, appear in the dielectric
spectrum at the same temperature range. This explains why the
γ-process becomes almost invisible in the dielectric spectra of
dx.doi.org/10.1021/mp400116q | Mol. Pharmaceutics 2013, 10, 3612−3627
Molecular Pharmaceutics
Figure 11. Dielectric spectra of IND samples obtained by cryogenic grinding (panels a and c) and room temperature milling (panels b and d). The
dielectric data were collected above and below the glass transition temperature.
Figure 12. Comparison of dielectric spectra measured at the same
temperature T = 216 K for anhydrous IND obtained by vitrification
and hydrated samples prepared using cryomilling and ball milling.
the cryomilled and ball-milled IND samples. The only evidence
for the existence of this γ-mode is the poorly noticeable high
frequency flank of this process visible in Figure 11d. Therefore,
the question arises: What is the molecular origin of the υ-mode?
As it was mentioned in the previous part of this paper the only
difference between the milled and vitrified IND samples is the
water content. While the quench-cooled sample is anhydrous,
the cryomilled and ball-milled materials contain small amounts
of water. Thus, one can suppose that the υ-process is related to
the mobility of water molecules present in milled IND. In order
to characterize the υ-modes observed for both water-containing
IND materials, the υ-relaxation times have been calculated as
the inverse of the frequency of the maximum peak position, i.e.
τ = 1/2πf max. As clearly seen in Figure 13 the temperature
dependences of log τυ, determined for the amorphous samples
obtained by various mechanical treatments, overlay each other.
This result is not surprising in the light of the data displayed in
Figure 12, where the maximum of υ-modes for both water-
3623
Article
Figure 13. Temperature dependence of α (diamond symbols), υ (solid
and crossed circle symbols), γ (open symbols), relaxation times
determined for anhydrous IND and both examined hydrated samples.
Solid lines indicate VFT and Arrhenius fits to the α-relaxation times
and secondary γ-, and υ-relaxation times, respectively.
containing IND samples are exactly the same at 216 K. Since the
relaxation times of the υ-process plotted as an inverse of
temperature exhibit the Arrhenius behavior from fitting of eq 8
to the experimental data, the activation energy of the υ-mode was
determined to be 52.4 ± 0.3 kJ/mol. It is worth noting that this
value is in good agreement with the activation energy of water
relaxation found for other water mixtures such as e.g. DPG + H2O
(43 kJ/mol),69 or hydrated lidocaine HCl (49 kJ/mol).70 Moreover
it is also close to the value of Ea for supercooled water examined in
some confined water systems (Ea ≈ 44.4 ± 3.7 kJ/mol).71
As it was demonstrated above, the effect of water on the
molecular dynamics of IND in the glassy state is significant.
dx.doi.org/10.1021/mp400116q | Mol. Pharmaceutics 2013, 10, 3612−3627
Molecular Pharmaceutics Article

Table 3. Apparent Solubility of Crystalline and Amorphous Forms of IND Obtained by Vitrification, Ball Milling and
Cryomilling Determined after 24 h of the Experiment
solubility of indapamide mean value n = 9
amorphous form
crystalline form vitrified ball milled cryomilled
media temp [°C] mg/L RSD [%] mg/L RSD [%] mg/L RSD [%] mg/L RSD [%]
H2O 25 81 2.55 96 2.50 107 2.30 100 2.60
37 109 2.40 125 1.90 144 0.70 144 1.38
0.1 M HCl 25 88 2.63 102 2.50 125 2.20 122 3.10
37 106 2.25 140 2.50 149 1.00 151 1.23
phosphate buffer pH 6.8 25 81 2.92 91 2.10 103 2.70 96 2.30
37 106 2.57 120 2.10 136 1.50 140 0.40

Thus, the question is: How does water af fect the structural
relaxation process and consequently the values of glass transition
temperature and f ragility of the examined systems? In the
literature one can find abundance of experimental data showing
that water is a common plasticizer and an increase of water
content leads to a drop of Tg.68 To verify this statement for
IND, we analyzed the dielectric spectra of cryomilled and room
temperature milled samples collected above Tg. As illustrated in
panels a and b of Figure 11, in the supercooled region of the
milled materials the well resolved structural relaxation process
is observed. Moreover, in both cases the maximum of the α-
peak appears in our frequency window at the same temperature
conditions as for the quenched IND sample. Additionally,
comparison of the shape and position of the structural
relaxation processes collected at the same temperature for all
examined materials reveals only one difference between
dielectric spectra. Namely, the contribution of the dc Figure 14. Dynamic solubility studies of crystalline and amorphous
conductivity is more pronounced in the case of ball-milled (vitrified) indapamide (n = 3).
IND than for the melt-quenched and cryomilled samples. Thus,
one can expect the same temperature dependences of τα for all greater concentration of the drug in comparison to that of the
examined IND materials. Indeed, as depicted in Figure 13, crystalline material (Figure 14). Following the supersaturation
regardless of the amorphization method, the α-process detected peak, concentration of indapamide decreased to 129.4 ± 4.3 mg/L
in the dielectric loss spectra of IND exhibits the same pattern of at 10 min of the experiment. The drop in the drug concentration
behavior. Consequently, the values of the glass transition was related to solution mediated crystallization of the amorphous
temperature and dynamic fragility of IND do not depend on phase to indapamide hemihydrate, however the drug levels in
the applied amorphization technique. This result is consistent solution remained significantly higher (p < 0.05) than the “steady-
with the DSC thermograms, which show the characteristic state” concentration for crystalline indapamide hemihydrate. From
signature for the Tg in the heat flow with an onset at 377 K for Table 3 it can be seen that irrespective of the amorphization
each examined IND sample (see inset in Figure 2). technique, in each medium, the apparent solubility of amorphous
Part C: Solubility Advantage from an Amorphous IND IND, determined at 24 h of the experiment, is 13% to 42% greater
Drug. As a final point we would like to present results of than that of the crystalline form. The greatest value (151 mg/L i.e.
apparent solubility measurements of crystalline and all examined 42% higher than for crystalline IND) was obtained for the
amorphous forms of indapamide. The obtained values are cryomilled sample in 0.1 M hydrochloric acid at 37 °C.
collected in Table 3. As a reference, crystalline IND was examined. Additionally, it was found that there was only a slight difference
Crystalline indapamide subjected to nonsink dissolution studies at between apparent solubility of the cryomilled and ball-milled
37 °C dissolved rapidly to a concentration of 106.8 ± 12.1 mg/L samples. The solubility of IND samples was determined with high
within the first minute of the experiment. Solubility of crystalline precision. The values of the relative standard deviation (RSD),
indapamide reached a plateau at 109.2 ± 9.6 mg/L within 30 min. calculated on the basis of nine replicates for each sample, were in
the range of 0.4−3.1%.

PXRD analysis confirmed that the solid residue recovered at the
end of the experiment was indapamide hemihydrate, indicating
that this crystalline form was physically stable and did not convert
in water to other forms of indapamide. Additionally, the solubility
of crystalline IND in water was found to be equal to 81 mg/L
when the experiment temperature was decreased to 25 °C. These
results are in good agreement with the literature data (75 mg/L).72
On the other hand, amorphous indapamide, obtained using
vitrification, reached a concentration of 394.6 ± 24.0 mg/L within
the first two minutes of the experiment, resulting in over 3-fold
3624
■
CONCLUSIONS
From our studies on amorphous IND we can draw the
following conclusions:
1. Quench-cooling of the melt, cryogenic grinding and
room temperature milling can be successfully applied to
produce IND in the amorphous state. Especially
important for commercial application seems to be
cryogenic milling, which was found to be very effective.
dx.doi.org/10.1021/mp400116q | Mol. Pharmaceutics 2013, 10, 3612−3627
Molecular Pharmaceutics
NMR and HPLC measurements combined with mass
spectrometry have shown that all examined amorphous
samples were of purity satisfying the strict pharmaceutical
requirements.
2. Investigations by means of FT-IR in a wide temperature range
have shown that conversion to the glassy state of IND leads to
an amide−imidic acid tautomerism, which is a reversible
proton transfer between different locations in the molecule.
Interestingly, the less stable imidic acid form of IND appears
independent of the applied amorphization technique.
3. Using BDS spectroscopy, we have studied the molecular
dynamics of vitrified and milled IND in the supercooled
liquid and glassy states. In all cases, at a given temperature, the
position as well as shape of the structural relaxation process
was found to be the same (the only difference between the
spectra was the higher contribution of dc conductivity in the
case of the ball-milled sample). Thus, there was also no
difference between the temperature dependence of structural
relaxation times determined for all examined samples. The
glass transition temperature calculated on the basis of BDS
measurements was found to be equal to 374 K (τα = 100 s)
for the vitrified and milled materials, and it is in good
agreement with the value determined from calorimetric
measurements (TgDSC = 377 K).
On the other hand, the molecular dynamics of milled
and melt-quench samples below Tg differ from each other
significantly. In all cases, the dielectric measurements reveal
only one, well-pronounced secondary relaxation process,
however its molecular origin is completely different. In the
case of the vitrified sample it was labeled as γ-mode. This
relaxation process with a low value of the activation energy
(34 kJ/mol) was found to be due to rotation of the
sulfonamide group. Also, for milled samples the υ-relaxation
with Ea = 54 kJ/mol, related to mobility of water molecules,
was observed. Additionally, for each examined sample, a
slower secondary mode (β), identified as a hidden JG
relaxation, which originates from intermolecular interac-
tions, was visible in the dielectric spectra.
4. It has been presented, using BDS, DSC and XRD
techniques, that amorphous indapamide prepared by
quench-cooling, cryogrinding and ball grinding is a
physically stable system in a wide temperature range,
above as well as below Tg. We did not notice any signs of
crystallization even one year after amorphization (storage
conditions: T = 293 K, humidity 10%).
5. Amorphous IND crystallizes in aqueous media to indapamide
hemihydrate. The supersaturation concentration of amor-
phous IND undergoing liquid-mediated crystallization at 37
°C in water was found to be nearly four times greater than the
equilibrium solubility of crystalline IND. It was found that,
irrespective of the amorphization technique, the apparent
solubility of amorphous indapamide was greater by 13−42%
than that of the crystalline form. The greatest value (151 mg/
L i.e. 42% greater than for crystalline IND) was obtained for
cryomilled sample in 0.1 M hydrochloric acid at 37 °C at 24 h
of the apparent solubility studies.
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: zaneta.wojnarowska@us.edu.pl.
Notes
The authors declare no competing financial interest.
3625
■
Article
ACKNOWLEDGMENTS
The authors Z.W., K.G., M.P. and W.S. are deeply grateful for
the financial support by the National Science Centre within the
framework of the Opus3 project (Grant No. DEC-2012/05/B/
1127NZ3/03233). L.T. and K.J.P. wish to acknowledge funding
for this research from Solid State Pharmaceutical Cluster
(SSPC), supported by Science Foundation Ireland under Grant
No. 07/ 1130SRC/B1158. Z.W. acknowledges the financial
assistance from FNP START (2013). M.D. is thankful for
support from PL-Grid Infrastructure.
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