ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2018, Vol. 92, No. 1, pp. 84–92. © Pleiades Publishing, Ltd.

, 2018.

PHYSICAL CHEMISTRY
OF SOLUTIONS

Solubility of Gliclazide and Ion-Molecular Interactions

with Aminopropanol in Aqueous Solutions1
Imad I. Hamdan*, Dina El-Sabawi, and Rana Abu-Dahab
Faculty of Pharmacy, The University of Jordan, Amman, 11942 Jordan
*e-mail: I.hamdan@ju.edu.jo
Received September 19, 2016

Abstract—A new salt of gliclazide (GZD) was prepared and was shown to have a significantly higher aqueous
solubility at physiological pH together with superior dissolution profiles in comparison to GZD employing
an organic amino-alcohol base. Characterization by NMR, IR, DSC, conductometry and HPLC techniques
concluded that an ion pair salt is formed between acidic GZD and basic aminopropanol (AMP). In addition
to the presence of about 5% tightly bound water, hydrogen bonds appeared to form extensively between GZD,
AMP and water molecules. Unlike many of solubility enhancing approaches, the salt did not hamper the per-
meability of GZD as shown by transport through Caco-2 cells model. In vivo studies on rats confirmed that
the blood glucose lowering effect of GZD-AMP was significantly higher and more rapid compared to parent
GZD indicating an enhanced overall performance of the prepared salt.

Keywords: solubility, dissolution, gliclazide, Caco-2, salt, aminopropanol

DOI: 10.1134/S0036024418010107

1. INTRODUCTION improved dissolution, yet that might not be paralleled

with improved bioavailability because of potential
Poor aqueous solubility of drugs is a major cause
for low oral bioavailability. According to the BSC clas- decrease in permeability as a consequence of the forma-
sification system, the aqueous solubility of Class II tion large un-absorbable inclusion complex [16].
drugs is the major determinant of their oral bioavail- In a previous report, sodium salt of GZD was pre-
ability [1, 2]. Gliclazide (GZD) is a widely used oral
hypoglycemic compound that is known for its low pared and shown to offer significant improvement in
aqueous solubility. GZD (Fig. 1) is an acidic com- terms of dissolution rate [17]. Since the nature of the
pound with a pKα value of 5.8 but it also contains a salt (counter ion) is known to influence the physico-
weekly basic amino group that is likely to exist in ion- chemical properties and consequently pharmaceutical
ized form below the pH of about 2 [3–5]. Thus the behavior of the drug [18], we attempted to examine the
compound is predominantly in the rather lipophilic performance of aminopropanol (AMP) as an organic
non ionized form within the anticipated pH range of amino-alcohol base with the potential to form a salt
the stomach and some parts of the intestines. As a drug with GZD. The prepared salt (GZD-AMP) demon-
used for controlling blood sugar, rapid action is usually strated significantly higher solubilities and dissolution
a critical added advantage. However, high aqueous profiles while maintaining reasonable permeation
solubility and dissolution is usually essential if a rapid through Caco-2 cells. Furthermore, experiments on
action of the drug is to be attained.
rats suggested enhanced bioavailability of GZD if
Extensive efforts have been made to improve the administered as GZD-AMP.
solubility of GZD in water including preparing the
drug in a soft gelatin form [6], utilizing solid disper-
sion, and co-grinding technologies [7–11]. Some suc- 11
cess was also reported with micronization, micelliza- 10 12
tion and cyclodextrin inclusion complex formation O O O 89
5
[12–15]. However, some of the aforementioned tech- 4 S N 10' 11'
6 N 2 N
niques suffer disadvantages such as impracticality and 9'
H1 H
high cost. In case of cyclodextrin for example, although H3C 5' 3
the produced complex would undoubtedly demonstrate 6'

1 The article is published in the original.

Fig. 1. Chemical structure of gliclazide (GZD).

84
 SOLUBILITY OF GLICLAZIDE
2. MATERIAL AND METHOD
2.1. Materials
GZD that is 99.1% pure (by HPLC) was a gift from
Pharma International (Amman, Jordan). Potassium
dihydrogen phosphate and aminopropanol were
obtained from Sigma-Aldrich (Germany).
2.2. Preparation of GZD-AMP
16.15 g of GZD were dissolved in 200 mL of meth-
anol after which 3.75 g of aminopropanol dissolved in
50 mL of distilled water were added, mixed well and
the excess solvent was evaporated using a rotary evap-
orator with temperature maintained at 50°C. The
obtained residue was let to dry in a desiccator.
2.3. HPLC and Spectral Characterization of GZD-AMP
50 mg of the salt were dissolved in 50 mL methanol
and further diluted 10 times using phosphate buffer
pH 4.5, before being injected onto HPLC system. The
content of GZD was calculated using a similarly pre-
pared calibration curve of GZD.
Untreated GZD and GZD-AMP were subjected to
differential scanning calorimetric analyses using a
Mettler Toledo calorimeter (Mettler, Toledo
DSC823e, Switzerland) configured to a Mettler® Star
software system (Mettler, Toledo, Switzerland). FTIR
spectra were recorded using a Shimadzu® FTIR spec-
trometer (Shimadzu® 8400S IR spectrophotometer,
Japan) using the KBr disk method. 1H NMR spectra
were recorded using a 350 MHz Bruker NMR spec-
trometer.
2.4. Potentiometric and Conductometric Titrations
Samples of GZD-AMP (~350 mg) were dissolved
in 50 mL of distilled water and titrated potentiometri-
cally with a standardized 0.1 M HCl. The obtained salt
was also assessed by measuring the conductivity of
solutions of GZD-AMP and for comparison the con-
ductivity values of AMP alone were also measured in
similarly prepared solutions.
2.5. Solubility Studies
Solubility studies were conducted in different
media: water, HCl (pH 1.2) and phosphate buffers
having pH values of 4.5 and 6.8 and the dissolved
amount was quantified by HPLC. The HPLC system
was comprised of a UV detector and a pump (Merck-
Hitachi, Tokyo-Japan). The employed chromato-
graphic conditions were based on a previously pub-
lished method [19]. In brief, a reversed phase C18 col-
umn was employed (5 μm, 200 × 4.6 mm i.d., Thermo
Scientific, USA). The mobile phase consisted of a
mixture containing 40% acetonitrile and 60% of
25 mM phosphate buffer at pH 3.5 and was run at a
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 92
85
flow rate of 2 mL/min. The monitoring wavelength
was set at 235 nm.
2.6. Apparent Partition Coefficient (logPapp)
Determination
Octanol/water apparent partition coefficient was
determined as log(coctanol/cwater) for both untreated
GZD and GZD-AMP salt using 10 mg of the solid
material in aqueous buffer solutions (pH 1.2, 4.5, or
6.8). The concentrations of untreated GZD or its salt
were determined in the aqueous and organic phases
using HPLC. Partition coefficient experiments were
conducted in triplicate.
2.7. Dissolution Tests
Comparative dissolution experiments of untreated
GZD and GZD-AMP were carried out using a Copley
Scientific dissolution apparatus, DIS6000 (Copley,
UK). The tests were performed according to pharma-
copoeial specifications using Apparatus 2 (paddle
method). The three media employed for testing were
0.1 M HCl, phosphate buffer pH 4.5 and phosphate
buffer pH 6.8. Paddle rotation was set at 75 rpm.
Medium temperature was set at 37 ± 0.5°C. Samples
(2 mL) were withdrawn at pre-determined time points
(10, 20, 30, 45, 60, and 120 min). Percentage release of
GZD in each case was determined using the HPLC
method described above.
2.8. Caco-2 Permeation Study
Caco-2 cells were incubated at 37°C in an atmo-
sphere of 5% CO2 and were routinely maintained by
replacing the culture media with fresh one every 2–
3 days by regular passage.
In order to determine the highest safe concentra-
tion of GZD and GZD-AMP, an MTT proliferation
assay was performed. For transport experiment, Caco-
2 cells with an average cell density of 1 × 105 cells/cm2
were seeded onto 12-well trans-well plates and were
allowed to grow for 21 days until confluent monolayers
were obtained. Culture media was changed every 2–
3 days. On the day of experiment, culture media was
aspirated and both sides of the monolayers were
washed three times and pre-incubated with PBS then
solutions of test compounds (GZD and GZD-AMP)
were added to the donor compartment at a concentra-
tion equivalent to 12.5 μg/mL of GZD in PBS, while
the receiver chambers were filled with fresh PBS. The
volumes of solutions in the apical (AP) and basolateral
(BL) chambers were 500 and 1500 μL, respectively.
Samples (150 μL) were taken from the receiver com-
partments at 30, 60, 120, and 240 min time points. At
the last time point (240 min), samples were taken from
the donor compartments as well in order to confirm
mass balance. Samples were stored at –20°C until
HPLC analysis (n = 3).
No. 1 2018
86 HAMDAN et al.
2.9. In vivo Study
Pharmacological effects (pharmacodynamic) of
GZD and its potential salt were measured using oral
glucose loading model in rats and employing blood
glucose level (BGL) as a pharmacodynamic marker
[20]. In vivo studies on animals were performed in the
animal house of the Applied Sciences University
(Amman, Jordan) and in accordance with the interna-
tionally accepted code of ethics pertaining animal
handling. Twelve healthy rats weighing between 240–
270 g were included in this study and divided into two
groups of six (A) and (B). The rats were fasted over-
night, and then group (A) was given 0.8 mL of GZD
suspension (25 mg/kg), 0.8 mL of suspension base,
and 1 g/kg of glucose solution. Group (B) on the other
hand, was given 0.8 mL of GZD-AMP solution,
0.8 mL of suspension base, and 1 g/kg of glucose. In
all cases administration was carried out via oral intu-
bation. During the experiment the rats were abstained
from food and water. Blood samples were collected
from rats tails and glucose levels were checked using
ACCU-CHEK Performa® blood glucose meter after
0, 20, 40, 70, 110, 170, 230, 290, 350, and 440 min.
3. RESULTS AND DISCUSSION
3.1. Characterization of GZD-AMP Salt
The composition of the obtained substance (GZD-
AMP salt) was assessed by HPLC in addition to deter-
mination of water content. Karl Fischer titration indi-
cated a water content of 6.3%. According to HPLC
data the percentage of GZD in GZD-AMP was 77.4%
which accords with a salt formed between GZD and
AMP in a ratio of 1 : 1 and containing 5.4% of water.
DSC thermograms of GZD and the obtained salts
are shown in Fig. 2. While GZD exhibited a clear
sharp endothermic peak indicating a melting point of
171°C, the thermogram of GZD-AMP exhibited a
rather unique behavior in the sense that the broad
endothermic peak which most likely belongs to evap-
oration of water (dehydration) had its maximum at
145°C reflecting its very strong binding to drug mole-
cules. Overlapping with the dehydration peak are two
small sharp peaks at 138 and 145°C that most likely
belong to the melting of the salt and suggesting the
presence of two forms of molecular arrangements,
which one of them might have developed while the so
closely bound water molecules were leaving the struc-
ture. That was supported by the manually measured
melting point where values in the range of 125–136°C
were obtained. Moreover, a broad exothermic peak
was observed with its maximum at 185°C for GZD-
AMP which most likely reflects recrystallization and
molecular rearrangement processes as a consequence
of initial melting and loss of the tightly bound water
molecules. It was interesting to obtain a melting point
for the salt less than that of the free acidic drug because
that would indicate looser binding of salt molecules to
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
each other. It is highly likely that the apparently tightly
bound water molecules to the salt structure contribute
substantially to this effect.
The FTIR spectrum of GZD (Fig. 3) was consis-
tent with that previously reported in literature [7, 21–
24]. The FTIR spectra for GZD-AMP showed some
chemical shifts in comparison to GZD (Fig. 3). The
most obvious shifts were those of carbonyl group
(1709.5 to 1601 cm–1), sulfonyl group bands (1350 to
1291 cm–1; 1164 to 1174 cm–1), aromatic bands (919 to
876 cm–1) and to some extent those of the amide N‒H
bonds (3273 to 3269 cm–1; 2948 to 2940 cm–1). Two
absorption bands at 3191.7 and 3112.7 cm–1 that most
likely correspond to the acidic sulfonamide N‒H were
almost completely lost in the spectrum of GZD-AMP,
which supports the assumption that the sulfonamide
proton is attached to the basic amino group of AMP.
Presence of significant percentage of water was evident
by the very broad band covering the area above
3000 cm–1 in the spectrum of GZD-AMP. It is note-
worthy that most of the observed shifts occurred
towards lower frequency indicating weakening of the
original relevant bonds as a result of strong hydrogen
bond formation. Thus the observed results accords
with possible ion pair formation between GZD and
AMP with the affected groups (N–H, C=O and sulfo-
nyl) being greatly involved in hydrogen bond forma-
tion between donors and acceptors of different GZD
molecules on one hand and those of AMP and the
closely attached water molecules on the other hand.
Moreover π−π interaction between the aromatic rings
of GZD molecules seemed to be encouraged in the salt
form as evident by the significant shifts in the charac-
teristic absorption bands of the aromatic ring.
NMR spectra for GZD were in very good agree-
ment with those reported in literature [25], while those
for GZD-AMP exhibited significant shifts that reflect
serious changes in the way which GZD molecules
were arranged. Table 1 summarizes the chemical shifts
for both GZD and GZD-AMP obtained in DMSO.
The most obvious changes were those corresponding
to the replaceable protons of the amid (N3-H) and
sulfonamide groups (N1-H) where the latter disap-
peared completely which is in support of the involve-
ment of the acidic sulfonamide proton in salt forma-
tion with the basic amino group of AMP. The reso-
nance for N3-H was dramatically shifted to lower
frequency (7.7 to 6.7) suggesting a parallel decrease in
acidity of that proton and richer electronic density in
its environment when the salt is formed. That accords
with loss of sulfonamide proton to AMP where the
acquired negative charge on N1 enriches the sur-
rounding environment with electrons, potentially
through resonance involving the carbonyl group and
extending to the proton on N3 making it more electron
rich and consequently resonating at a lower frequency.
Aromatic protons H5, 5' and H6, 6' were also
clearly shifted to lower frequency which suggests inter-
Vol. 92 No. 1 2018
 SOLUBILITY OF GLICLAZIDE
(a)
2 mW
(b)
20 mW
50 100
0 5
Fig. 2. DSC thermograms for GZD-AMP (a) and GZD (b).
action of the aromatic rings of adjacent GZD mole-
cules with each other through pi-stacking [26, 27].
Water appeared as a distinctly broad band in the spec-
trum of GZD-AMP at δ 4.95 which is significantly
higher than δ 3.3 that is expected for water in DMSO
[28], which supports the DSC-based conclusions in
the sense that water was tightly bound to the structure
of salt through hydrogen bonding.
Overall, the obtained data support the formation of
salt with consequent changes in the attraction forces
between the drug molecules (apparently getting
weaker) due to tight binding of GZD to both of water
and AMP molecules through hydrogen bond forma-
tion with donor and acceptor groups on the three mol-
ecules (GZD, AMP, and water). The seemingly week
intermolecular association of GZD molecules along
with the strong binding to water molecules was
reflected in the clearly lower melting point obtained by
DSC in addition to the clearly high temperature of
dehydration (145°C).
An aqueous solution (10 mM) of the obtained salt
exhibited a basic pH value (~8.1) that is most likely
due to the inherent basic behavior of AMP which may
dissociate from GZD in aqueous solutions. In order to
gain insight about the dissociation of the presumed
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
87
150 200 250
10 15 20
salt in aqueous media, the conductivity of different
solutions with increasing concentrations of the salt
was measured. A plot of obtained conductivity mea-
sures versus concentration is presented in Fig. 4. It is
clear from Fig. 4 that while the profile of AMP alone
showed a change in slope at about 1 mM, no such
change was observed for GZD-AMP. AMP, as an
amino alcohol, was reported to form unique patterns
of intramolecular hydrogen bonds O‒H···N that links
the alcohol and amino terminals together with a ten-
dency to form aggregates at high concentrations [29,
30]. Thus, it could be postulated that higher concen-
trations of GZD-AMP encourage intermolecular
hydrogen bonding on the account of the intramolecu-
lar ones (for AMP) with a subsequent dimer (or higher
order of aggregates) formation. Due to seemingly
heavy involvement of the amino and hydroxyl groups
in strong hydrogen bonds between GZD and AMP in
the resulting salt, no sufficient intermolecular hydro-
gen bonds could be formed with the consequent
inability to form molecular aggregates e.g. dimers and
trimers. Accordingly it is quite likely that a substantial
percentage of GZD-AMP may not dissociate but the
solubilization of GZD is achieved as a result of added
polar groups from AMP.
Vol. 92 No. 1 2018
88 HAMDAN et al.

100

90

1041,60
(a)

469,57
80

1242,26
2361,59

705,83
634,79
751,75
70

2835,41

1309,42
1284,36

896,83
812,48
60

2867,15

1596,57
T, %

3191,72
3112,74

996,47
50

40

2948,51

1087,64

532,09
919,60

667,90
3273,17

30

1350,15
20

1435,79

1164,37
10

1709,57
95

90

706,10
85 (b)

776,23
80

813,70
850,45
75
2361,76

1352,11
1384,97

876,59
70
T, %

65
1174,32

549,78
60
670,98
1088,38
1528,51

55
2863,23

1291,52

50
1241,85
1601,75
3269,00
3416,12

2940,83

1127,41

45

4000 3500 3000 2500 2000 1500 1000 500

Wavenumbers, cm−1

Fig. 3. FTIR spectra for GZD (a) and GZD-AMP (b).

When a solution of the salt was titrated with HCl a were in good agreement with that provided by HPLC
rather typical weak base titration curve was obtained analysis, DSC and NMR.
with one jump suggesting 1 : 1 reaction with HCl. Cal-
culations based on the observed potentiometric end
point indicated a GZD percentage within the salt 3.2. Solubility and Partition Coefficient
equal to 74% and a percentage of GZD-AMP close to The obtained values for solubility and apparent
93.5% i.e., 6.5% of the total weight corresponds to partition coefficients (expressed as logP) are shown in
bound water. Thus the results of titration experiment Table 2. In comparison to GZD, the solubility of

RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 92 No. 1 2018

 SOLUBILITY OF GLICLAZIDE 89

Table 1. Detailed 1H NMR assignments of resonances in cant role in the solubilization of the whole ion pair
the spectra of GZD and GZD-AMP. Literature values for which accords with the DSC, NMR, conductometric
GZD based on reference [25] are shown in bold between and FTIR findings that were discussed earlier. More
brackets evidence perhaps can be deducted from the observa-
Proton GZD GZD AMP tion that the partition coefficient did not increase pro-
portionally as the pH dropped from 6.8 to 1.2; which
H of N1 9.6b (9.36) Disappeared* reflects that even at low pH values where dissociation
H of N3 7.7–8.1 (7.72) 6.7* is not complete, no parallel amount of GZD moved to
H5 7.83 (7.81) 7.61* the organic phase. Will this increasingly high polarity
of the obtained salt favorably influence the dissolu-
H6 7.38 (7.38) 7.15* tion, absorption and bioavailability of the drug? Or it
C-Methyl protons 2.36 (2.41) 2.34 may hinder the absorption as the drug moiety became
H9ax 2.30 (2.29) d.o.h too hydrophilic? Further explorations were made on
H9eq 2.90 (2.85) d.o.h dissolution and in vivo effect of the prepared salt in the
following sections.
H10 2.49 (2.51) d.o.h
H11ax 1.57 (1.65) 1.67
H11eq 1.393 (1.33) 1.33 3.3. Dissolution Studies
H12ax 1.57 (1.34) 1.67 The obtained dissolution profiles in the three
H12eq 1.39 (1.52) 1.33 media tested are shown in Fig. 5. At the three pH val-
ues examined GZD-AMP exhibited a superior perfor-
d.o.h, the peak disappeared or got hidden underneath other peaks mance compared to GZD free acid. At low pH value
for AMP. of 1.2 GZD-AMP did not release more than 20%, yet
b Broad.
that was more than triple of the amount released for
* Indicates resonances that are mostly affected by salt formation. GZD free acid (~ 5%). It was interesting to obtain such
an improvement in dissolution of GZD-AMP at the
GZD-AMP appeared to have almost doubled at very acidic pH (1.2) despite its observed lower solubil-
pH 4.5 while appeared to have decreased at pH 1.2. At ity. The obvious higher hydration of GZD-AMP as a
pH 6.8 however, a very dramatic increase in solubility result of the tightly bound water molecules may
was observed i.e., almost thousand times explain the observed faster dissolution despite lower
equilibrium solubility. The most obvious difference in
(25705 μg/mL) the solubility of native GZD which
the % release of the two was observed at pH 4.5 where
was about five times higher than a previously reported
released GZD-AMP was about 8 times higher than
value for a GZD sodium salt [17]. Thus, the observed GZD free acid through the 2 h period of the test. At
very high aqueous solubility was difficult to explain in pH 6.8, the initial release was much faster for GZD-
terms of simple ion dissociation. It is likely that the AMP compared to GZD, but towards the end of dis-
highly hydrophilic substituents of AMP play a signifi- solution period the two appeared to release almost
100% of GZD. That was consistent with GZD being
an acid with a pKα of 5.8, where it became extensively
Conductivity, µS/cm
280 ionized at pH 6.8 leading to significantly improved
solubility and dissolution [3, 4].
230
3.4. Caco-2 Permeation Study
180 The obtained cumulative percentage of absorbed
drug against time profile for each of GZD and GZD-
AMP is shown in Fig. 6. No substantial differences
130 appeared to exist between the extent of absorption of
GZD from its salt or free acid forms. However, GZD-
AMP was at least absorbed as much as GZD, if not
80
GZD-AMP slightly higher. As such this is expected to be an
improvement over GZD because while GZD-AMP
30 AMP exhibits almost similar permeability to GZD it also has
a higher solubility profile. The importance of solubil-
0.002 0.004 0.006 0.008 ity-permeability interplay when developing solubility
−20 Molar concentration enhancing formulations has been clearly demon-
strated by Dahan and Miller [16]. Solubility enhanc-
Fig. 4. Conductivity profiles for each of AMP alone (m) ing techniques such as cyclodextrins inclusion com-
and GZD-AMP (d). plexes, use of surfactants and polyethylene glycol have

RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 92 No. 1 2018

90 HAMDAN et al.

Table 2. Average solubility and partition coefficient data obtained for GZD-AMP in comparison to published data for
GZD (n = 3, RSD less than 5%)
GZD (literature values) GZD-AMP
Medium pH
solubility (μg/mL) apparent logP solubility (μg/mL) apparent logP
1.2 (HCl) 124a – 60 –0.36
4.5 (Phosphate buffer) 40a; 57b – 85 –0.71
6.8 (Phosphate buffer) 182a; 295b – 0.27c; 1.97d 25705 –1.4
a
[17]; b[31]; c[32]; d[12].

been shown to hinder the permeation of some drugs
such as carbamazepine, progesterone and hydrocorti-
sone [31–33]. Therefore GZD-AMP is expected to
exhibit an enhanced in vivo performance compared to
GZD as the former has a similar permeability profile
but with superior dissolution profiles.
3.5. In vivo Animal Study
For the sake of comparison between the bioavail-
ability of GZD and its prepared amino salt, the phar-
macodynamic effect of GZD on blood glucose level
(BGL) was taken as a measure of the absorbed drug
[34]. Plots of average BGL against time for each of

120
(a)
100
Released, %

80
60
40
20
0
20 40 60 80 100 120 140
120 Time, min
(b)
100
Released, %

80
60
40
20
0
20 40 60 80 100 120 140
120 Time, min
(c)
100
Released, %

80
60
40
20
0
20 40 60 80 100 120 140
Time, min

Fig. 5. Dissolution profiles for GZD (s) and GZD AMP (d) in HCl (a), phosphate buffer at pH 4.5 (b) and phosphate buffer at
pH 6.8 (с). RSD for six determinations was always less than 7%.

RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 92 No. 1 2018

 SOLUBILITY OF GLICLAZIDE
50
45
40
Cumulative percentage
35
30
25
20
15
10
5 Time, min
0 1 2 3 4 5
Time, h
Fig. 6. Cumulative percentage of transported GZD (s)
and GZD-AMP (d) from apical to basolateral side as a
function of time.
GZD and GZD-AMP compared to control were
obtained and presented in Fig. 7. Accordingly, BGL
was most seriously altered within the first two hours of
dosing. In the control group where animals received
no treatment BGL appeared to be the highest, whereas
the lowest BGL was achieved by GZD-AMP. It is
noteworthy that GZD-AMP appeared to work so rap-
idly that there was no period of BGL rise although the
rats were administered a high dose of sugar at the time
of dosing. On the other hand, a period of BGL rise
that lasted for 0.5–1 h was observed for both GZD and
control before it started to decline to even hypoglyce-
mic levels (Fig. 7). At many time points in the course
of the test, the difference between GZD and GZD-
AMP mounted to more than 20%. In order to enable
statistical comparison between the groups, the areas
under the curve for BGL versus time profiles were cal-
culated using trapezoid method for each animal.
Using one way ANOVA, the differences between the
means were found to be statistically significant at α =
0.05, where F, p, and F critical values were 4.22, 0.035,
and 3.68, respectively. Therefore, GZD-AMP has
been shown to be a salt of GZD with favorable phar-
maceutical properties over GZD in terms of solubility,
dissolution rates and in vivo bioavailability. The
apparent near 20% improvement in the bioavailability
and pharmacodynamic effect of GZD-AMP in com-
parison to GZD could be seriously needed for some
patients who take the hypoglycemic drug with meals
seeking rapid action. The promising data collected in
this work encourages further investigations in which
larger number of animals is employed and perhaps
clinical studies on humans where the drug itself might
be measured in plasma instead of the indirect pharma-
codynamic assessments.
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
91
120
100
Blood glucose level
80
60
40
20
0
100 200 300 400
Fig. 7. Plot of blood glucose level against time for GZD
(s), GZD-AMP (d), and control (m).
CONCLUSION
A salt of GZD with the organic amino-alcohol base
(aminopropanol) has been prepared and characterized
in vitro and in vivo. NMR, IR and DSC alongside
other analytical data supported the formation of the
salt between acidic GZD and AMP with heavy
involvement of hydrogen bonds between the polar
groups of the two molecules. Conductometric titra-
tions suggest the maintenance of the ion pair formed
between GZD and AMP and solubilization of the drug
may be achieved through hydrogen bonding between
water molecules and the ion pair as a single unit. The
salt was shown to have an enhanced dissolution profile
over native GZD with better or at least similar perme-
ation profile using caco-2 cell model system. Experi-
ments on animals demonstrated faster absorption of
GZD-AMP and lower blood glucose levels compared
to parent GZD. Thus, unlike other solubility enhanc-
ing approaches, the prepared salt was shown not to
negatively affect the permeability of the drug.
ACKNOWLEDGMENTS
The authors wish to thank the Deanship of Aca-
demic Research at The University of Jordan for finan-
cial support (grant recommendation no. 88/2012–
2013).
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