Solid state and solubility study of a potential anticancer drug-drug molecular salt of diclofenac and metformin

Article abstract of DOI:10.1016/j.molstruc.2021.130166

To improve the physicochemical properties of diclofenac (DFA) and exert its potential anti-tumor effect in combined pharmacotherapy of metformin (MET), a new drug-drug molecular salt of DFA and MET (DFA-MET) is formed and characterized. The single-crystal X-ray diffraction analysis shows that DFA-MET is a three-dimensional (3D) supramolecular structure constructed by different hydrogen-bonding interactions between DFA and MET with 1:1 stoichiometry by proton transfer reaction. In addition, Hirshfeld Surface analysis shows that both the hydrogen-bonding interactions and the Vander Waals maintain the 3D supramolecular structure of DFA-MET together. Compared with pure DFA and pure diclofenac sodium (NaDFA), the dissolving behavior and permeability of DFA-MET by forming drug-drug molecular salt in physiological pH environments are enhanced remarkably.

Full text of DOI:10.1016/j.molstruc.2021.130166

Journal of Molecular Structure 1234 (2021) 130166
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Research paper
Solid state and solubility study of a potential anticancer drug-drug
molecular salt of diclofenac and metformin
Wen-Quan Fenga,∗, Ling-Yang Wang a, Jie Gaoa, Ming-Yu Zhaoa, Yan-Tuan Lia,b,∗
,
Zhi-Yong Wua, Cui-Wei Yan a
a School of Medicine and Pharmacy, and College of Marine Life Science, Ocean University of China, Qingdao, Shandong 266003, PR China
b Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, 266003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To improve the physicochemical properties of diclofenac (DFA) and exert its potential anti-tumor effect in
combined pharmacotherapy of metformin (MET), a new drug-drug molecular salt of DFA and MET (DFA-
MET) is formed and characterized. The single-crystal X-ray diffraction analysis shows that DFA-MET is
a three-dimensional (3D) supramolecular structure constructed by different hydrogen-bonding interac-
tions between DFA and MET with 1:1 stoichiometry by proton transfer reaction. In addition, Hirshfeld
Surface analysis shows that both the hydrogen-bonding interactions and the Vander Waals maintain the
3D supramolecular structure of DFA-MET together. Compared with pure DFA and pure diclofenac sodium
(NaDFA), the dissolving behavior and permeability of DFA-MET by forming drug-drug molecular salt in
physiological pH environments are enhanced remarkably.
Received 28 December 2020
Revised 9 February 2021
Accepted 12 February 2021
Available online 26 February 2021
Keywords:
Diclofenac
Metformin
Drug-drug molecular salt
Anticancer
Published by Elsevier B.V.
Hirshfeld surface analysis
CCDC 2,052,795 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cam-
bridge Crystallographic Data centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: +441,223 336,033).
found both in vitro and in vivo to inhibit angiogenesis and to in-
duce apoptosis in tumors [8,9]. However, the poor solubility lim-
its its drug effect and anti-tumor activity better display [10]. So,
a way to improve solubility of NSAIDs has practical implications.
The salification strategy, one of the crystal engineering, is a good
way of solving this problem and is widely used in improving the
physicochemical properties of APIs without changing pharmacolog-
ical behaviors [11,12]. Therefore, it is a valuable way to increase the
dissolvability of NSAIDs in water by means of using the salification
strategy, and particularly optimize the anti-tumor effect of NSAIDs.
A lot of researches indicated that diclofenac (DFA, Fig. 1(a))
has the anticancer activity like other NSAIDs [13]. For example,
it is reported to suppress growth and migration of tumors, espe-
cially human glioblastoma cells [13,14]. But DFA is considered a
class II agent in the Biopharmaceutical Classification System (BCS)
and present poor solubility and bioavailability like most NSAIDs
[15]. Therefore, it is important to find an effective way to improve
physicochemical properties and bioavailability of DFA. Thanks to
the simplicity and availability of salification, this is an obvious and
effectual way to improve poor aqueous solubility, low bioavailabil-
ity and antitumor activity of DFA [16–19].
1. Introduction
Cancer is one of the most health-threatening diseases in the
world, and tens of thousands of lives are taken from us be-
cause of the malignant tumor in the past couple of decades.
To find an effective medicine in treating cancer becomes an ur-
gent to solve the problem in the pharmaceutical industry [1,2].
Though a number of drugs to fight cancer have entered the market
recently, an increasingly urgent need to seek new drugs is raised,
due to an increase in incidence and drug resistance [3–5]. Signif-
icantly, it is very important to study that reforming the chemical
structure of existing drugs and discovering anti-tumor activity be-
cause of high costs, long cycle and big risk of drug development,
especially in clinical trial [6].
Recently, a growing body of research have shown that Nons-
teroidal Antiinflammatory Drugs (NSAIDs) may play a part in the
prevention and treatment of many types of cancer [7]. NSAIDs are
There are reports of salinization of DFA, such as diclofenac
sodium (NaDFA, Fig. 1(c)) [20]. But its absorption is rapid after oral
administration with a half-life of 1–2 h, requires sustained drug re-
lease [21]. This requires that the solubility of salinization of DFA
in this experiment is greater than that of pure DFA and less than
that of NaDFA. With the development of salinization strategy, it is
∗
Corresponding authors.
E-mail address: 1413295162@qq.com (W.-Q. Feng).
https://doi.org/10.1016/j.molstruc.2021.130166
0022-2860/Published by Elsevier B.V.

W.-Q. Feng, L.-Y. Wang, J. Gao et al.
Journal of Molecular Structure 1234 (2021) 130166
Fig. 1. The chemical structures of (a) DFA, (b) MET and (c) NaDFA used in this study.
Table 1
of significance for choice of conjugated acid/base, particularly anti-
tumor compounds, to achieve goals of drug combinations [16–19].
Metformin (MET, Fig. 1(b)) typically in the form of hydrochloride,
can lower blood sugar and is still a first-line drug for the treat-
ment of type 2 diabetes mellitus (T2DM) [22]. Recently researchers
are increasingly finding that metformin use can be associated with
a reduced risk of cancer, and it is also possible that metformin
is working more directly on the tumor process [23–25]. Another
study suggests that DFA in combination with MET can inhibit mi-
gration and proliferation in human glioma cells, and reduce the
level of cellular lactate caused by MET [26]. Meanwhile DFA also
reduces pain of patients caused by tumors [27]. Over all, it plays
an important part for improvements in the low solubility and the
anti-tumor activity of drug itself to achieve a combination of DFA-
MET by means of salinization technology.
Crystal data and structure refinement for DFA-
MET.
Empirical formula
C18 H22N6O2Cl2
Formula weight
Temperature/K
Crystal system
Space group
425.31
296(2)
monoclinic
P21/c
˚
a/A
8.8755(9)
10.1824(10)
22.402(2)
90
˚
b/A
˚
c/A
α/°
β/°
γ /°
94.214(3)
90
3
˚
Volume/A
2019.1(3)
4
Z
ρ
calcg/cm3
1.399
μ/mm-1
0.349
Based on the way of thinking, we successfully prepare a drug-
drug salt through proton exchange reaction between DFA and MET.
The structure and preliminary physicochemical properties of DFA-
MET are characterized by single-crystal X-ray diffraction, PXRD,
FTIR, DSC and pharmacokinetic studies. Especially, Hirshfeld Sur-
face analysis is used to analysis the different kinds of interactions
between DFA and MET in the drug-drug salt. Our results show that
MET had been dramatically enhance the water solubility and pen-
etrability of DFA in the drug-drug molecular salts and effectively
improved its pharmacokinetics. We believe that results obtained
in this study will be valuable in the development of new dosage
forms.
F(000)
888.0
Crystal size/mm3
Radiation
0.335 × 0.102 × 0.098
MoKα
2ꢀ range for data
collection/°
Index ranges
Reflections
(λ = 0.71073)
6.078 to 55.154
-11 ≤ h ≤ 11,
-13 ≤ k ≤ 13,
-29 ≤ l ≤ 29
31,305
collected
Independent
reflections
4663 [R
Data/restraints/parameters= 0.0804, R
int
sigma
Goodness-of-fit on
F2
= 0.0701]
4663/0/319
1.021
Final R indexes
[I>=2σ (I)]
Final R indexes [all
data]
R1 = 0.0554,
wR2 = 0.1046
R1 = 0.1256,
wR2 = 0.1262
0.26/−0.24
2. Materials and methods
Largest diff.
−3
˚
2.1. Materials
peak/hole / e A
Diclofenac (DFA) and Metformin HCL were purchased from
Shanghai Aladdin Bio-Chem Technology Co., LTD. All solvents used
in this study were analytical grade and obtained from local suppli-
ers.
solid was obtained by filtration and then dried under vacuum for
24 h. Yield: 35.1 mg, 82.6%.
2.2.2. Single crystal X-ray diffraction (SCXR0D)
SCXRD data for DFA-MET were collected on a Rigaku Saturn
2.2. Methods
˚
CCD diffractometer with Mo-Kα radiation (λ = 0.71073 A) at
296 K, and processed with the SAINT software. The crystal struc-
ture of DFA-MET was solved using SHELXT by direct methods and
refined for non-H atoms using SHELXL by full-matrix least-squares
on F2 with anisotropic displacement parameters [28]. H atoms con-
nected to C atoms were fixed in geometrically constrained posi-
tions. H atoms connected to O and N atoms were included in the
located positions. The crystallographic data and refinement details
are given in Table 1.
2.2.1. Preparation of DFA-MET
First of all, Metformin HCl (30 mmol, 5 g) reacted with NaOH
(30 mmol, 1.2076 g) in water (20 mL) to prepare MET free base
(about 2 h). Then ethanol (80 mL) was added to make the MET
free base dissolve in ethanol, and the resulting NaCl was insol-
uble in ethanol, so as to achieve the purpose of separating the
MET free base. The ethanol was removed by rotary evaporation
to obtain MET free base during drying. Yield: 4.54 g, 73.2%. Fi-
nally, Equimolar amounts of DFA (0.1 mmol, 29.6 mg)and MET free
base (0.1 mmol, 12.9 mg) were dissolved in a methanol-acetonitrile
mixture (1:4 v:v) and stirred at room temperature. The resulting
clear solution was filtered and allowed to evaporate slowly. The
2.2.3. Hirshfeld surface analysis
The Hirshfeld surface and their 2D fingerprint plots are indica-
tive for the qualitative and quantitative analysis of intermolecular
2

W.-Q. Feng, L.-Y. Wang, J. Gao et al.
Journal of Molecular Structure 1234 (2021) 130166
interactions in the crystal. They were produced by the Crystal Ex-
plorer software, using the calculated crystal structure parameters
[29]. dnorm, di, and de surface were mapped over a fixed color
˚
˚
˚
scale of −0.552−1.359 A, 1.000−2.838 A, and 0.773−2.809 A, re-
spectively [30].
2.2.4. Powder X-ray diffraction (XRPD)
PXRD was performed using a Bruker D8 Advance diffractometer,
˚
using a Cu-Kα (λ = 1.54178 A) source at 40 kV and 400 mA. Each
sample was collected from 5 ° to 50 ° (2θ) at ambient temperature
and scan speeds of 2 ° min−1, And the results are analyzed by Jade
5.0 software.
2.2.5. Fourier transform infrared spectroscopy (FT-IR)
Fig. 2. ORTEP view of the cocrystal with the atom displacement ellispoids drawn
at a 30% probability level, showing the atom numbering. H atoms are drawn as
spheres of arbitrary radii.
The FT-IR spectra of samples were recorded using a Nicolet
model Impact 470 Fourier transform infrared spectrometer and KBr
as pellets. The KBr pellet was used to obtain background spectra.
The range was set from 4000 to 4000 cm−1
.
2.3. Results and discussion
2.2.6. Differential scanning calorimetry (DSC) and thermogravimetric
analysis (TGA)
2.3.1. SCXRD analysis
Single crystal X-ray diffraction reveals DFA-MET crystallized
in the monoclinic P21/c space group. As the Fig. 2 shows, one
molecule of DFA and one molecule of MET constitute the asym-
metric unit connected by N − H•••O hydrogen bond involving an
amidogen of the MET and a carboxylate Ion of the DFA contain-
ing an intramolecular hydrogen bond. The details of the hydrogen
bonds are given in Table 2. As shown in Fig. 2, two molecules of
MET are interconnected by N − H•••O hydrogen bonds to form
the centrosymmetric dimer described as R22(8) in graph set no-
tation. And each of these connects an adjacent molecule of DFA
through the R22(8) system and is connected to another a molecule
of DFA N − H•••O hydrogen bond, which form a one-dimensional
(1D) chain. The 1D chains are further connected through R24(8)
(Fig. 3) to form a two-dimensional (2D) layer (Fig. 4). Finally, a lot
of 2D layers repeatedly pile up to form a three-dimensional (3D)
supramolecular structure along a axis by various weak interactions
(Fig. 5).
DSC and TGA measurement of DFA-MET were carried out em-
ploying a Mettler Toledo TGA/SDTA 851e module and a Mettler
Toledo DSC 822e module, respectively. The samples tested were
heated from 50 to 400 °C at a scanning rate of 5 °C min−1 under
a N2 flow of 20 mL min−1
.
2.2.7. Solubility experiments
The solubility of DFA and DFA-MET in the pH 1.2 pH, pH4.0
and pH6.8 buffer solutions at 37.0
0.5 °C was measured by the
shake-flask method. An excess of each sample was placed in an Ep-
pendorf tube, 6 mL of the solvent followed was added and stirred.
After 48 h, the suspension was filtered through a 0.22-μm nylon
filter, and the filtrate was later determined by a Cary 300 spec-
trophotometer at a detection wavelength using a validated analyt-
ical method [31]. The experimental methods were repeated three
times.
2.2.8. Intrinsic dissolution rate (IDR) experiments
Intrinsic dissolution rate (IDR) measurements were carried out
on a RC-6 dissolution tester by the rotating disk method. About
100 mg of pure DFA and DFA-MET were compressed for 1 min at
2.5 ton per inch2 to form the 8-mm-diameter of a nonporous disk
covered by paraffin wax and leaving a flat surface to measure the
dissolution. Then these disks were rotated at 100 rpm in 400 mL
2.4. Hirshfeld surface analysis
The Hirshfeld surface and their 2D fingerprint plots anal-
ysis are effective tools for studying crystal packing. The sin-
gle molecule of DFA in crystal is used as the input for calcu-
lations. Through analysis of this results, four red spots, where
close contacts are formed, are found on the dnorm surfaces of
the single molecule of DFA, which means that there are not the
strong charge-assisted hydrogen bonds between each molecule
of DFA. As shown in Fig. 6, the red spots on the dnorm sur-
medium preheated to 37
0.5 °C at different pH values, respec-
tively. 5 mL of dissolution medium was withdrawn at regular inter-
vals, the same volume of the corresponding buffer solution added
to the original medium. Finally, the concentration of all the sam-
ples filtered through a 0.22-μm nylon filter were measured by UV
analysis and repeated three times.
face correspond to the close contacts due to four N − H•••
O
hydrogen-bonding interactions [29]. As shown in Fig. 7, the dom-
inate surface contacts for DFA in DFA-MET can be split five
ways: H•••H (37.3%), H•••C/C•••H(18.8%), H•••Cl/Cl•••H(17.4%),
H•••O/O•••H(14.3%) and H•••N/N•••H(3.4%). The most of the con-
tributions over the total Hirshfeld surface are H•••H (37.3%) con-
tacts, suggesting that the molecular surface is composed of a sea
of H atoms. A large amount of H atoms over the surface sup-
ported H•••C contacts to be the second one (14.8%). In addition,
the third largest population of contacts is attributed to the H•••Cl
contacts, which shows that the weak hydrogen-bonding interac-
tions are also the major driving forces between 3D supramolecular
structure. In contrast, H•••O strong hydrogen-bonding interactions
account for only 14.3% of total Hirshfeld surface, showing that the
strong hydrogen-bonding interactions are not the main intermolec-
ular forces for the crystal packing. Finally, H•••N contacts play only
a small part in the crystal packing.
2.2.9. Permeability experiments
Permeability experiments of DFA and DFA-MET were measured
by the modified Franz diffusion cell apparatus through a cellulose
nitrate membrane (0.45 mm, Sartorius, Germany). The membrane
was placed in between the donor compartment and the recipient
compartment to which 5 mL of buffer medium (pH 6.8) was added.
After the buffer medium was kept at 37 0.5 °C and rotated at
50 5 rpm. About 20 mg powder samples were placed on the
membrane. The sample solution (0.5 mL) was extracted from the
receptor chamber every 1 h and replaced with the same volume of
buffer medium every 1 h over 8 h. Finally, the concentration of DFA
and DFA-MET were measured by UV analysis from the respective
calibration plots. The experimental methods were repeated three
times.
3

W.-Q. Feng, L.-Y. Wang, J. Gao et al.
Journal of Molecular Structure 1234 (2021) 130166
Table 2
˚
Hydrogen-Bonding Geometries for DFA-MET (A, °).
•••
•••
•••
•••
A
D-H
A
d(D-H)
d(H
A)
d(D
A)
ࢬD-H
•••
•••
N(1)-H(1)
N(1)-H(1)
Cl(2)
O(1)
0.84
0.84
0.87
0.87
0.87
0.91
0.81
0.86
0.96
0.96
2.48
2.33
2.04
2.06
2.22
1.99
2.13
2.57
2.34
2.81
2.9660
2.9838
2.8341
2.9239
3.0828
2.8961
2.9313
2.9417
2.7415
3.7565
118
138
152
171
171
174
171
108
105
167
•••
N(2)-H(2A)
O(2)
N(2)-
a
•••
H(2B)
N(3)-
H(3A)
O(2)
b
•••
N(3)-H(3B)
N(4)
•••
O(1)
N(5)-
c
•••
N(5)-H(5B)
C(17)-
H(5A)
O(1)
•••
N(2)
•••
H(17C)
N(4)
C(18)-
d
•••
H(18B)
Cl(1)
Symmetry codes: (a) 2-x,1-y,-z (b) 2-x,2-y,-z (c) 1-x,1-y,-z (d) 1-x,1/2 + y,1/2-z.
Fig. 3. A one-dimensional (1D) chain composed of DFA and MET and hydrogen bond between them.
Fig. 4. A two-dimensional (2D) layered structure composed of DFA and MET.
2.5. XRPD analysis
18.82 °, 20.52 °, 24.38 °, 28.46 ° and 17.7 °, 22.52 °, 23.36 °, 28.34
°, 29.74 °, 35.84 °, respectively. Furthermore, the experimental pat-
tern of DFA-MET is found in congruence with the simulated pat-
tern from the data of single-crystal X-ray, demonstrating the crys-
tallinity and high purity of DFA-MET samples.
As an effective way of characterization, XRPD is widely ap-
plied in the detection of phase transition and purity of crystalline
phases. As shown in Fig. 8, the simulated pattern of DFA-MET is
compared to the experimental patterns of DFA-MET, pure DFA, and
pure MET. The results show that a striking diffraction pattern of
DFA-MET makes difference between those of pure DFA and pure
MET, demonstrating the formation of a new solid phase . There are
some obvious characteristic diffraction peaks at 2θ=11.74 °, 13.6
°, 15.06 °, 19.4 °, 20.06 °, 23.62 ° and 26.7 ° While characteristic
diffraction peaks of DFA and MET are 2θ=10.7 °, 13.44 °, 15.14 °,
2.6. FT-IR analysis
The FT-IR spectra of MET, DFA and DFA-MET are presented in
Fig. 9. The spectrum of pure DFA shows a distinctive band at 3322
cm−1, representing stretching of -NH of DFA. Whereas the corre-
sponding bands of DFA-MET are shown at 3446 cm−1, which is in-
4

W.-Q. Feng, L.-Y. Wang, J. Gao et al.
Journal of Molecular Structure 1234 (2021) 130166
Fig. 5. A three-dimensional (3D) supramolecular structure of the DFA-MET, each color represents a asymmetric unit.
ferred that the intermolecular hydrogen bonds between DFA and
MET cause the shift in the absorption spectra. On the other hand,
the pure DFA also have a distinctive peak corresponding to C = O
stretching vibration at 1693 cm−1. But the corresponding peak of
DFA-MET is observed at 1650 cm−1 and shifts to lower wave num-
bers, demonstrating that the carboxylic acid transform into the car-
boxylate Ion by interacting with DFA and MET. In addition, the dis-
appearance of broad band of carboxylic group at about 3100–2500
cm−1 in the spectrum of DFA-MET similarly backs it up.
2.7. TGA-DSC analysis
Two thermal curves of DFA-MET are shown in Fig. 10, respec-
tively. The DSC curve (blue line) indicates that there is only one
major endothermic peak at 202.71 °C, which reflects to the melt-
Fig. 6. Interactions of DFA and MET with the generated Hirshfeld surface in the
salt.
˚
Fig. 7. 2D fingerprint plots according to the dnorm value (−0.929~1.430 A), Hirshfeld surface view about these interactions, and the relative contributions of various inter-
•••
•••
H, (d) Cl
•••
•••
H, (f) N
•••
H.
actions for whole crystal are shown for (a) total, (b) H
H, (c) C
H, (e) O
5

W.-Q. Feng, L.-Y. Wang, J. Gao et al.
Journal of Molecular Structure 1234 (2021) 130166
Fig. 8. XRPD comparison of simulated pattern for (a) DFA-MET, experimental pattern for (b) DFA-MET, (c) DFA, and (d) MET.
Fig. 9. The FT-IR spectra of (a) MET, (b) DFA and (c) DFA-MET.
Fig. 10. DSC (blue line) and TGA (red line) curves of DFA-MET recorded at
−1
•
5 °C min heating rate.
ing point of DFA-MET. As it is seen, this melting point differentiates
melting points of pure DFA (156–158 °C), NaDFA (283–285 °C) and
pure MET (223–226 °C), revealing the formation of new phases,
and the increase in stability probably due to stronger intermolecu-
lar interactions (including hydrogen bonds) between DFA and MET.
Additionally, the result shows that there is almost no other en-
dothermic peak except the main one, which further illustrates this
salt relatively stable and does not exist solid-solid phase transi-
tion or polymorph transformation. Meanwhile, TGA curve (red line)
shows that there is no apparent the phenomena of weight loss be-
fore decompose of DFA-MET, showing that DFA-MET does not con-
tain any solvate molecules, which is qualitatively consistent with
the above result of SCXRD.
relevant pH (pH 1.2, pH 4.0 and pH 6.8). The solubility data at pH
1.2, pH 4.0 and pH6.8 are shown in Table 3. It turns out that the
solubility of DFA, NaDFA and DFA-MET increase with the increasing
the pH value. And it is worth noting that the solubility of DFA-MET
increase at pH 1.2, pH 4.0 and pH6.8 by 20 times,377 times and
108 times more than its API, respectively. The uptake of oral ad-
ministered drugs primarily occurs in the small intestine whose pH
correspond to pH 6.8. The solubility of DFA-MET increase greatly,
which may lay the foundation for favorable bioavailability. In addi-
tion, the solubility of the DFA-MET at pH 6.8 is one-tenth that of
NaDFA, and is not high enough to achieve the sustained release of
NaDFA. The observations that overall solubilities of DFA-MET are
higher than that of pure ACA in the three pH and that of pure
NaDFA in the two pH can mainly describe as a result of the follow-
ing reasons. the incorporation of DFA with high soluble MET into
the crystal lattice of the cocrystal can drive the increase in the sol-
ubility of DFA, because the aqueous solubility of MET (303 mM) is
much higher than that of DFA.
2.8. Solubility studies
It is the ultimate goal of cocrystallization for most APIs to im-
prove their solubility, as it usually increases the bioavailability of
drug compounds. Therefore, the excess amounts of solids in sus-
pension have been used to study the dissolution behavior of DFA,
NaDFA and DFA-MET in aqueous solutions with pharmaceutically
6

W.-Q. Feng, L.-Y. Wang, J. Gao et al.
Journal of Molecular Structure 1234 (2021) 130166
Table 3
The solubility of DFA-MET, DFA and NaDFA in pH 1.2, 4.0, and 6.8 buffer medium.
Solubility (mM)
Solubility medium
DFA-MET
DFA
NaDFA
pH 1.2 buffer
pH 4.0 buffer
pH 6.8 buffer
0.0921 0.0012
1.91 0.06
0.00669 0.00026
0.00726 0.00015
0.0376 0.0021
0.00259 0.00029
0.0175 0.0017
2.84 0.02
38.5
0.1
Fig. 12. Flux of DFA-MET, DFA and NaDFA vs. time.
Fig. 11. IDR profiles of DFA-MET and DFA at pH 1.2, 4.0, and 6.8.
Table 4
IDR values of DFA-MET and DFA in pH 1.2, 4.0, and 6.8 buffer medium.
IDR (mg min−1 cm−2
DFA-MET
)
Solubility medium
DFA
pH 1.2 buffer
pH 4.0 buffer
pH 6.8 buffer
0.166 0.012
0.471 0.039
0.564 0.007
0.00922 0.00035
0.0119 0.0002
0.0155 0.0014
2.9. Intrinsic dissolution rate (IDR) studies
In order to further investigate the kinetic solubility of DFA-MET,
the experiments of IDR were conducted in pH 1.2, 4.0, and 6.8
buffer solutions. The intrinsic dissolution rate of DFA-MET com-
pared with that of pure DFA and the calculated IDR values at pH
1.2, pH 4.0 and pH 6.8 are shown in Fig. 11 and Table 4, respec-
tively. The results show that the dissolution rate of DFA-MET is
found to 10-fold to 60-fold faster than that of the pure DFA in
three buffer solutions, and the IDR values of both DFA-MET and
DFA increases with the increase of pH value. Overall, comparing
with the pure DFA, it not only has a higher IDR, but also shows
a greater advantage in solubility, suggesting that DFA-MET can en-
hance pharmacokinetics in vivo and bioavailability of DFA. In terms
of the structure, the higher intrinsic dissolution rate of the DFA-
MET could be explained that the MET, due to its very high solu-
bility, are drawn out of the crystal lattice into the aqueous phase,
leading to the formation of the incompact clusters of the DFA.
Fig. 13. Cumulative amount of DFA-MET, DFA and NaDFA permeated vs. time plot.
pure NaDFA are shown in Fig. 12. As the Fig. 12 shows, com-
pared with pure DFA and pure NaDFA, the permeate flux of DFA-
MET moves up sharply for the first hour, and then reaches a sta-
ble state controlled by membrane diffusion dynamics. Addition-
ally, as shown in Fig. 13, the cumulative diffused amounts of DFA-
MET, pure DFA and NaDFA have a significant positive correlation
with penetration time. And more notably, DFA-MET presents better
permeability than pure DFA and pure NaDFA from the beginning.
The cumulative diffused amount of DFA-MET is 2.4854 mg/cm2 at
8 h, equivalent to 13.97 times that of DFA (0.1779 mg/cm2) and
48.41times that of NaDFA (0.05134 mg/cm2). It shows the sodium
salt of DFA, NaDFA, does not increase the permeability of DFA. In-
stead, it decreases the permeability, which can be understood as
the price of increased solubility of DFA. The membrane diffusion
2.10. Permeability studies
Permeability as an important physicochemical property of drugs
influences the process of absorption and delivery, and provides a
good pharmacokinetic behavior of drugs. In this experiment, the
diffusion behaviors of DFA-MET, pure DFA and pure NaDFA are
measured at a pH 6.8 buffer medium every 1 h for 8 times. The
flux and cumulative diffused amounts for DFA-MET, pure DFA and
7

W.-Q. Feng, L.-Y. Wang, J. Gao et al.
Journal of Molecular Structure 1234 (2021) 130166
capacity of molecular salt formed by DFA and MET is obviously
higher than that of pure DFA and pure NaDFA, and it shows that
the solubility and permeability of DFA are improved by forming
molecular salt, which is good for the bioavailability of DFA.
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3. Conclusions
In this work, a new drug-drug molecular salt of DFA and MET
was successfully obtained using solution crystallization method
and characterized by means of all kinds of analyze method. The
crystal structure showed that the molecules of DFA was directly
connected to the molecules of MET by hydrogen bonds, and inter-
actions between molecules of DFA compared with the dimer struc-
ture of DFA was too small, leading to the improved physicochemi-
cal properties of DFA. Hirshfeld Surface analysis indicated that the
hydrogen-bonding interactions between DFA and MET accounted
for just half of weak interactions, the other half were almost Van-
der Waals. In addition, the dissolving behavior and permeability of
DFA-MET by salt formation in physiological pH environments were
enhanced dramatically. To sum up, it is better to improve their
physical and chemical properties through salt formation to achieve
potential synergistic anticancer effect.
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Declaration of Competing Interest
All authors declare that No conflict of interest exists.
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We thank Shandong Province Key Research and Development
Project (2018GSF118174), Qingdao People’s Livelihood Science and
Technology Project (18–6–1–95-nsh), and the NSFC-Shandong Joint
Fund for Marine Science Research Centers (grant no U1606403)
and the National Natural Science Foundation of China.
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