Role of Hydrogen Bonds in Formation of Co-amorphous
Valsartan/Nicotinamide Compositions of High Solubility and Durability
with Anti-hypertension and Anti-COVID-19 Potential
Introduction Valsartan
(VAL) is an anti-hypertensive drug belonging to the angiotensin
II receptor blockers (ARBs), which according to the recommendations
of the European Cardiac Society (2016) provide alternative treatment
for patients intolerant of angiotensin-converting enzyme inhibitors
(ACE inhibitors) or aldosterone receptor blocker therapies. High blood pressure is one of the most important
risk factors for cardiovascular diseases, and hence, ARBs are an important
class of drugs decreasing the risk of heart diseases. However, ARBs are poorly water-soluble drugs, classified
according to the Biopharmaceutics Classification System (BCS) as class
II with high permeability and low solubility. The bioavailability of BSC class II drugs is limited by their solvation
rate and in the case of ARBs, it ranges from 13% (eprosartan) to 70%
(irbesartan), and for the title, VAL is 23%. No wonder that pharmaceutical
industry is intensively looking for solutions that will allow them
to reduce the dose of the drugs by enhancing solubility, which improves
their bioavailability. Several strategies have been used to improve
the oral bioavailability of VAL, such as co-crystallization, − formation of host–guest β-cyclodextrin complexes, , synthesis of microcapsules, nanoparticles, and polymeric solid dispersions. − It is worth noting that the solid dispersion strategy leading to
amorphization of pharmaceutical components is especially important
in the case of pharmaceuticals, such as VAL, which are difficult to
crystallize, and attempts to crystallize them usually lead to a sticky
solid, amorphous film, or loose powder. Generally, amorphous forms of drugs dissolve more rapidly
and are
better absorbed than the corresponding crystalline forms. Hence, amorphization gives an opportunity to
improve dissolution rates of drugs, which directly leads to the increase
in their bioavailability. The amorphous
solids (glasses) in contrast to the crystalline solids have no well-defined
molecular arrangement, but their enhanced molecular mobility leads
to a better solubility, providing the advantage to use this type of
active pharmaceutical ingredients (APIs). They possess higher free energy and entropy than the corresponding
crystals and tend to recrystallize to lower energy crystalline forms,
so that special methods, such as the solid dispersion strategy, are
necessary to stabilize the amorphous state. The solid dispersion is
defined as a dispersion of one or more active substances suspended
in an inert carrier and prepared by melting, dissolution, or melting–dissolution
techniques. To date, polymeric amorphous
dispersions containing the drug incorporated in a glass polymeric
matrix have commonly been used for stabilization of amorphous drugs.
However, such solid dispersions have several disadvantages, including
low miscibility necessitating large polymer/drug ratios and hygroscopicity. Therefore, a new alternative strategy, leading
to co-amorphous solid dispersions, in which the polymer was replaced
by a low-molecular weight substance, such as nicotinamide (NIC), has
been developed. These mixtures combine two or more low-molecular weight
components, called as co-formers, into a homogenous amorphous single
phase. Addition of a co-former gives
advantages, such as preventing recrystallization of the amorphous
API and preventing phase separation observed in polymeric matrices.
The solid dispersions can be prepared using milling (ball milling
and cryo-milling), solvent evaporation (rotary evaporation, spray
drying, and freeze drying), quench cooling, and hot-melt extrusion
techniques. Some of these techniques
have been applied to improve solubility
of VAL; however, polymer-based glass solutions were mainly obtained. − , − Mei et al.
reported on the co-amorphous mixture where amino acids were used as
low molecular excipients to increase the solubility of VAL up to 1000
times; however, some of these solid dispersions turned out to be unstable
after 3 months. Other solid dispersions
of VAL with small molecular co-formers have also been reported: (a)
with inorganic, alkalizing agents (CaCO 3 ) and (b) organic N -methyl- d -glucamine. A 46-fold and ninefold increase
in solubility, respectively, has been achieved. A co-amorphous system of VAL with vanillin, loaded in mesoporous
silica particles with an improved dissolution rate, has also been
described. However, the co-formers applied
in the abovementioned API-GRAS (GRAS—generally recognized as
safe, FDA list of safe food additives) systems do not contribute to
cardiovascular health effects. There are only two, recently described,
co-amorphous API–API systems containing VAL, obtained by Shastri
et al. , First, the VAL–clinidipine system
turned out to be stable under standardized accelerated conditions
(40 °C/75% RH) for 1 month and exhibited an enhanced dissolution
profile. The second system, VAL–nifedipine,
showed the same stability as the VAL–clinidipine system and
2.19-fold-increased solubility compared to the free VAL. The current paper is focused on co-amorphous
formulations, consisting
of VAL (as the API) and NIC, as pharmaceutically acceptable, small-molecular
weight excipient, which has been chosen in view of its high water
solubility and beneficial health effect. NIC, which is one of the
two components of vitamin B 3 (PP), exhibits several beneficial
health effects, among others: (1) supports the cardiovascular system
(promotes relaxation and reduces anxiety), (2) stimulates the immune
system ( vide infra ), (3) reduces inflammation [prevents
lung tissue damage and slows the progress of degenerative arthritis
( vide infra )]. Hence, it can be rationally used as
the excipient of the VAL composition. Both VAL and NIC contain several functional groups, which are part
of the relevant synthons needed to create strong intermolecular interactions,
for example, carboxyl, tetrazolyl, and amide groups (VAL) and amide
and pyridyl groups (NIC) . The Δp K a values
also indicate the possibility of creating VAL–NIC compositions
with strong intermolecular bonds. Based on a generally accepted “rule
of three”, the co-crystal formation (lack of proton transfer)
is expected for Δp K a < 0 and
the salt formation (proton transfer occurs) is expected for Δp K a > 3. The Δp K a values of the investigated VAL/NIC system
are <0; therefore, hydrogen bonding instead of salt formation is
expected .
Moreover, the 1:1 molar ratio (VAL/NIC) used fully corresponded to
the recommended daily dosages of VAL (80–320 mg, therapeutic
dose) and NIC (20–50 mg, preventive supplementation dose).
Therefore, this type of formulation can be used to treat hypertension
as an improved, more bioavailable, and dually acting composition. ARB drugs (e.g., VAL) target the renin–angiotensin–aldosteron
system by blocking angiotensin II type 1 receptors (AT1R). − Based on ClinicalTrials.gov resources, 57 clinical trials regarding the influence of ARBs in
the treatment of the COVID-19 disease have been commenced until October
2020. Five of these clinical trials are focused only on VAL and one
(ClinicalTrials ID: NCT04335786) proposes two mechanisms: (1) ARBs
block the excessive angiotensin-mediated AT1R activation; (2) ARBs
upregulate ACE2, which reduces angiotensin II concentrations and increases
the production of the protective vasodilator angiotensin-(1–7).
NIC, which is the second component of the solid dispersion, can also
be useful in the treatment of the COVID-19 disease. It has been shown
that NIC, apart from the beneficial influence on the cardiovascular
system, prevents lung tissue damage and
is a key compound that enhances immune response, especially in viral
infections. It has a strong anti-inflammatory
effect on respiratory lung damage, which is crucial for COVID-19 patient
treatment. , In this work, four co-amorphous
compositions of VAL and NIC were
synthesized by the solid- and solution-state protocols. Solid dispersions
and their components (VAL and NIC) have been studied by Fourier transform
infrared (FT-IR) spectroscopy supported by the calculations in the
framework of density functional theory (DFT), Bader’s quantum
theory of atoms in molecules (QTAIM), and the non-covalent interactions index (NCI) method, which can be treated as an extension of QTAIM.
It is a very useful tool for distinction and visualization of non-covalent
interactions of different types. Bader’s QTAIM has been widely
applied to study covalent and non-covalent interactions. − However, in the area of multicomponent pharmaceutical solid forms,
these calculations have rarely been used, despite the fact that non-covalent bonds play a crucial role in
the formation of co-crystals or co-amorphous solid dispersions and
hence in changing properties of these solid forms. , Here, we employed QTAIM and NCI to elucidate the strength and nature
of the possible non-covalent interactions occurring in VAL/NIC co-amorphous
solid dispersions and compared them with the interactions occurring
in single-component crystals. Additional calculations of solvation
free energy were utilized to investigate the interactions between
the solute (co-amorphous solid dispersions) and solvents (water and
ethanol). Other experimental techniques, such as solid-state NMR (ssNMR),
X-ray powder diffraction (XRPD), differential scanning calorimetry
(DSC), and scanning electron microscopy (SEM), have also been applied
to confirm amorphization of the obtained materials. The solubility
of co-amorphous formulations was investigated in water and phosphate
buffer (pH 7.4) and compared with the solubility of pure VAL. 

Materials and Methods 2.1 Materials VAL
was extracted from
Vanatex (Polpharma SA) and Axudan (Sandoz GmbH). The detailed procedure
and analyses are described in the Supporting Information . Briefly, VAL was extracted from tablets with acetonitrile and purified
by the slurry crystallization method developed by Mei et al. The structure of the API obtained was confirmed
by spectroscopic (NMR, MS, and IR), thermal (DSC and melting point)
and X-ray (XRPD) analyses (SM). NIC and solvents were purchased from
Sigma-Aldrich and used without further treatment. 2.2 Synthesis of Co-Amorphous Mixtures (1) VAL/NIC LAGs:
equimolar amounts (1 mmol) of the two components
(VAL and NIC) were ground using an agate mortar and pestle with addition
of six drops of ethanol per each 30 min with a total grinding time
of 30 min (VAL/NIC LAG 30 min) and 60 min (VAL/NIC LAG 60 min). The
resulted powder was dried and kept in a desiccator. (2) VAL/NIC slurry:
equimolar amounts (1 mmol) of the two components (VAL and NIC) were
added to 3 mL of ethanol and the resulted slurry was stirred and refluxed
at 70 °C for 3 h. The solvent was evaporated on a rotary vacuum
evaporator. The solid obtained was dried and stored in a desiccator.
(3) VAL/NIC ball mill: equimolar amounts (1 mmol) of the two components
(VAL and NIC) were placed in a stainless-steel 12 mL grinding jar
with 10 stainless-steel balls (4 mm diameter) and ground with addition
of 12 drops of ethanol at 450 rpm for 60 min. The obtained sticky
solid was dissolved in anhydrous ethanol, which was then evaporated
on a rotary vacuum evaporator. The solid obtained was dried for 30
min under vacuum (0.7 mbar) and stored in a desiccator. 2.3 Fourier Transform Infrared Spectroscopy The Fourier
transform mid infrared spectra of VAL and NIC and the
obtained co-amorphous mixtures were measured at 2 cm –1 resolution and by 32 scans on a Nicolet-Nexus spectrometer, in the
region of 4000–400 cm –1 , using the KBr pellet
technique. Vibrations of the essential groups, which may be involved
in the hydrogen bond formation, have carefully been analyzed. The
data were processed using Spectrum software. 2.4 DFT,
QTAIM, and NCI Calculations DFT calculations were performed
with the Gaussian 09 set of programs. The structures of components (VAL and NIC) and
two designed VAL/NIC heterodimers (VAL/NIC1 and VAL/NIC2) differing
in the existing supramolecular synthons were optimized and the IR
intensities were calculated with the hybrid functional B3LYP combined
with the 6-311++G(d,p) basis set. These two heterodimers (VAL/NIC1
and VAL/NIC2) were selected after preliminary calculations of five
different heterodimers using the semiempirical AM1 method, as the
two heterodimers with the lowest energy ( Figure S10 ). This approach allowed us to assess how the arrangement
of components (VAL and NIC) in the co-amorphous formulation affected
the shift of vibrations of essential functional groups involved in
the formation of hydrogen bonds. To compare predicted and observed
frequencies, the wavenumbers were scaled by 0.967. Potential energy distribution analysis was performed using
the FCART program. QTAIM and NCI calculations
were performed for the designed heterodimers with the structures optimized
at the B3LYP/6-311++G(d,p) level both in the gas phase and in ethanol.
Additionally, these calculations were conducted for VAL/VAL homodimers
and NIC/NIC homotrimers to investigate binding energies in individual
component crystals. Therefore, homodimers/trimers were cut from the
crystal structures of NIC (Cambridge Structural Database (CSD) refcode
NICOAM06) and VAL (CSD refcode KIPLIG), and the single-point DFT calculations
were performed. To investigate the intermolecular interactions between
ethanol and the investigated VAL/NIC system, these calculations were
also carried out for the designed solvated system (VAL/NIC1)EtOH and
crystal solvate of VAL cut from the crystal structure (CSD refcode
KIPLEC). The calculations in the framework of QTAIM were performed
with the use of Multiwfn (a multifunctional wavefunction analyzer)
software. It enabled us to find the bond
critical points (BCPs) of non-covalent interactions and to determine
the properties of electron density at these points, such as the electron
density at BCP (ρ BCP ), its Laplacian (∇ 2 ρ BCP ), the kinetic ( G BCP ), potential ( V BCP ), and total
( H BCP ) electron energy densities. The
energies of non-covalent interactions ( E bin ) were calculated from the V BCP values,
according to the equation in the form of E bin = V BCP /2, which was proposed by Espinosa
et al. To be able to state that a particular
non-covalent H···A interaction belongs to the group
of hydrogen bonds, we have used the first four criteria proposed by
Koch and Popelier (KP). According to
them, the hydrogen bond occurs when (i) the bond path between donor
(D) and acceptor (A) atoms exists, (ii) the ρ BCP values
lie in the range of 0.002–0.034 a.u., (iii) the ∇ 2 ρ BCP values lie in the range from 0.024 to
0.139 a.u., and (iv) a mutual penetration of the H and A atoms, which
is treated as “a necessary and sufficient” criterion,
exists (i.e., Δ r H = r H V – r H > Δ r A = r A V – r A and Δ r H + Δ r A >
0, where r H V and r A V are the van der Waals radii of
H and A atoms, respectively,
and r H and r A denote the distances between these atoms and BCP). Multiwfn software
was also used for calculations in the framework of the NCI method,
which can be treated as an extension of QTAIM theory. This method
is based on the relationship between the electron density (ρ)
and the reduced density gradient (RDG = [1/2(3π 2 ) 1/3 ][|∇ρ|/ρ 4/3 ]) and is a very
useful tool for the distinction and visualization of different types
of non-covalent interactions. The NCI calculations were visualized
with VMD (visual molecular dynamics) software and Gnuplot 5.2. Free energies
of solvation, its components (cavitation, dispersion,
non-electrostatic, electrostatic, and polarization energy), and dipole
moments were calculated for both heterodimers (VAL/NIC1 and VAL/NIC2)
and the solvated system (VAL/NIC1)EtOH with the polarizable continuum
model (PCM) with the integral equation formalism variant at the B3LYP/6-311++G(d,p)
level of theory with the Gaussian 09 package and compared with the
results obtained in the gas phase. The
solute cavity was created via overlapping of van
der Waals spheres, with water and ethanol as solvents. 2.5 Solid-State NMR The 13 C cross-polarization
magic angle spinning ( 13 C CP-MAS)
ssNMR experiments were performed on a Bruker Avance III 600 spectrometer
with resonance frequencies of 150.918 MHz. The spectra of components
and co-amorphous mixtures were collected with a spinning rate of 12
kHz, and the relaxation delays (RDs) during the acquisitions equaled
5 and 300 s. 2.6 X-ray Powder Diffraction XRPD data
were collected for VAL, NIC, VAL/NIC co-amorphous dispersions, and
VAL/NIC physical mixtures at room temperature with an Oxford Diffraction,
Xcalibur3 CCD diffractometer using Cu Kα radiation. XRPD patterns
were recorded for all samples after synthesis (1st day) and over 4th,
9th, and 11th months of storage under standardized conditions (25
°C, 60% RH)—for fully co-amorphized samples. XRPD experiments
were performed using transmission geometry at room temperature with
powdered samples sealed in capillary tubes and rotated about phi over
360° at 0.5°/S. CCD image data were processed using CrysAlisPRO
software. The obtained data were analyzed
using Fullprof software. 2.7 Differential Scanning Calorimetry DSC studies of VAL,
NIC, their physical mixture, and co-amorphous
mixtures were recorded using the Netzsch STA 409 C/CD instrument.
The samples were placed in sealed, non-hermetic aluminum pans and
scanned at a heating rate of 10.0 K/min under an argon atmosphere. 2.8 Scanning Electron Microscopy A VEGA3
TESCAN scanning electron microscope was used to collect photo-micrographs
of co-amorphous mixtures and their components. The samples were mounted
on a metal stub with adhesive tape and observed without coating under
high vacuum. For each sample, 200× magnification was applied. 2.9 Solution-State NMR Studies The 1 H NMR spectra were collected for co-amorphous formulations
in order to investigate contents of ethanol in the obtained formulations.
The 1 H NMR spectra were measured on a Bruker AV III 500
spectrometer in CDCl 3 with chemical shifts (δ) given
in ppm relative to TMS as an internal standard. 2.10 Solubility Studies The solubility
of the co-amorphous dispersions obtained and free VAL and physical
mixture of VAL and NIC was determined in distilled water and phosphate
buffer (pH = 7.4) at 37 °C to simulate physiological conditions.
The relevant sample was weighted in order to obtain supersaturated
solution in water/phosphate buffer using a 20 mL test tube immersed
in a thermostated water bath (37 °C) and equipped with a magnetic
stirrer. The contents of the test tube were continuously stirred for
15 min; then, an additional amount of the relevant solvent (e.g.,
0.1 mL) was added to the tube until the test substance completely
dissolved, as determined with the macrocamera. The experiment was
performed in triplicate and solubility values were expressed as a
mean ± standard deviation. The solubility values for solvated
co-amorphous solid dispersions and physical mixture were recalculated
in reference to pure VAL, taking into account the content of VAL relative
to NIC and EtOH in the obtained solvated solid dispersions. 

Materials VAL
was extracted from
Vanatex (Polpharma SA) and Axudan (Sandoz GmbH). The detailed procedure
and analyses are described in the Supporting Information . Briefly, VAL was extracted from tablets with acetonitrile and purified
by the slurry crystallization method developed by Mei et al. The structure of the API obtained was confirmed
by spectroscopic (NMR, MS, and IR), thermal (DSC and melting point)
and X-ray (XRPD) analyses (SM). NIC and solvents were purchased from
Sigma-Aldrich and used without further treatment. 

Synthesis of Co-Amorphous Mixtures (1) VAL/NIC LAGs:
equimolar amounts (1 mmol) of the two components
(VAL and NIC) were ground using an agate mortar and pestle with addition
of six drops of ethanol per each 30 min with a total grinding time
of 30 min (VAL/NIC LAG 30 min) and 60 min (VAL/NIC LAG 60 min). The
resulted powder was dried and kept in a desiccator. (2) VAL/NIC slurry:
equimolar amounts (1 mmol) of the two components (VAL and NIC) were
added to 3 mL of ethanol and the resulted slurry was stirred and refluxed
at 70 °C for 3 h. The solvent was evaporated on a rotary vacuum
evaporator. The solid obtained was dried and stored in a desiccator.
(3) VAL/NIC ball mill: equimolar amounts (1 mmol) of the two components
(VAL and NIC) were placed in a stainless-steel 12 mL grinding jar
with 10 stainless-steel balls (4 mm diameter) and ground with addition
of 12 drops of ethanol at 450 rpm for 60 min. The obtained sticky
solid was dissolved in anhydrous ethanol, which was then evaporated
on a rotary vacuum evaporator. The solid obtained was dried for 30
min under vacuum (0.7 mbar) and stored in a desiccator. 

Fourier Transform Infrared Spectroscopy The Fourier
transform mid infrared spectra of VAL and NIC and the
obtained co-amorphous mixtures were measured at 2 cm –1 resolution and by 32 scans on a Nicolet-Nexus spectrometer, in the
region of 4000–400 cm –1 , using the KBr pellet
technique. Vibrations of the essential groups, which may be involved
in the hydrogen bond formation, have carefully been analyzed. The
data were processed using Spectrum software. 

DFT,
QTAIM, and NCI Calculations DFT calculations were performed
with the Gaussian 09 set of programs. The structures of components (VAL and NIC) and
two designed VAL/NIC heterodimers (VAL/NIC1 and VAL/NIC2) differing
in the existing supramolecular synthons were optimized and the IR
intensities were calculated with the hybrid functional B3LYP combined
with the 6-311++G(d,p) basis set. These two heterodimers (VAL/NIC1
and VAL/NIC2) were selected after preliminary calculations of five
different heterodimers using the semiempirical AM1 method, as the
two heterodimers with the lowest energy ( Figure S10 ). This approach allowed us to assess how the arrangement
of components (VAL and NIC) in the co-amorphous formulation affected
the shift of vibrations of essential functional groups involved in
the formation of hydrogen bonds. To compare predicted and observed
frequencies, the wavenumbers were scaled by 0.967. Potential energy distribution analysis was performed using
the FCART program. QTAIM and NCI calculations
were performed for the designed heterodimers with the structures optimized
at the B3LYP/6-311++G(d,p) level both in the gas phase and in ethanol.
Additionally, these calculations were conducted for VAL/VAL homodimers
and NIC/NIC homotrimers to investigate binding energies in individual
component crystals. Therefore, homodimers/trimers were cut from the
crystal structures of NIC (Cambridge Structural Database (CSD) refcode
NICOAM06) and VAL (CSD refcode KIPLIG), and the single-point DFT calculations
were performed. To investigate the intermolecular interactions between
ethanol and the investigated VAL/NIC system, these calculations were
also carried out for the designed solvated system (VAL/NIC1)EtOH and
crystal solvate of VAL cut from the crystal structure (CSD refcode
KIPLEC). The calculations in the framework of QTAIM were performed
with the use of Multiwfn (a multifunctional wavefunction analyzer)
software. It enabled us to find the bond
critical points (BCPs) of non-covalent interactions and to determine
the properties of electron density at these points, such as the electron
density at BCP (ρ BCP ), its Laplacian (∇ 2 ρ BCP ), the kinetic ( G BCP ), potential ( V BCP ), and total
( H BCP ) electron energy densities. The
energies of non-covalent interactions ( E bin ) were calculated from the V BCP values,
according to the equation in the form of E bin = V BCP /2, which was proposed by Espinosa
et al. To be able to state that a particular
non-covalent H···A interaction belongs to the group
of hydrogen bonds, we have used the first four criteria proposed by
Koch and Popelier (KP). According to
them, the hydrogen bond occurs when (i) the bond path between donor
(D) and acceptor (A) atoms exists, (ii) the ρ BCP values
lie in the range of 0.002–0.034 a.u., (iii) the ∇ 2 ρ BCP values lie in the range from 0.024 to
0.139 a.u., and (iv) a mutual penetration of the H and A atoms, which
is treated as “a necessary and sufficient” criterion,
exists (i.e., Δ r H = r H V – r H > Δ r A = r A V – r A and Δ r H + Δ r A >
0, where r H V and r A V are the van der Waals radii of
H and A atoms, respectively,
and r H and r A denote the distances between these atoms and BCP). Multiwfn software
was also used for calculations in the framework of the NCI method,
which can be treated as an extension of QTAIM theory. This method
is based on the relationship between the electron density (ρ)
and the reduced density gradient (RDG = [1/2(3π 2 ) 1/3 ][|∇ρ|/ρ 4/3 ]) and is a very
useful tool for the distinction and visualization of different types
of non-covalent interactions. The NCI calculations were visualized
with VMD (visual molecular dynamics) software and Gnuplot 5.2. Free energies
of solvation, its components (cavitation, dispersion,
non-electrostatic, electrostatic, and polarization energy), and dipole
moments were calculated for both heterodimers (VAL/NIC1 and VAL/NIC2)
and the solvated system (VAL/NIC1)EtOH with the polarizable continuum
model (PCM) with the integral equation formalism variant at the B3LYP/6-311++G(d,p)
level of theory with the Gaussian 09 package and compared with the
results obtained in the gas phase. The
solute cavity was created via overlapping of van
der Waals spheres, with water and ethanol as solvents. 

Solid-State NMR The 13 C cross-polarization
magic angle spinning ( 13 C CP-MAS)
ssNMR experiments were performed on a Bruker Avance III 600 spectrometer
with resonance frequencies of 150.918 MHz. The spectra of components
and co-amorphous mixtures were collected with a spinning rate of 12
kHz, and the relaxation delays (RDs) during the acquisitions equaled
5 and 300 s. 

X-ray Powder Diffraction XRPD data
were collected for VAL, NIC, VAL/NIC co-amorphous dispersions, and
VAL/NIC physical mixtures at room temperature with an Oxford Diffraction,
Xcalibur3 CCD diffractometer using Cu Kα radiation. XRPD patterns
were recorded for all samples after synthesis (1st day) and over 4th,
9th, and 11th months of storage under standardized conditions (25
°C, 60% RH)—for fully co-amorphized samples. XRPD experiments
were performed using transmission geometry at room temperature with
powdered samples sealed in capillary tubes and rotated about phi over
360° at 0.5°/S. CCD image data were processed using CrysAlisPRO
software. The obtained data were analyzed
using Fullprof software. 

Differential Scanning Calorimetry DSC studies of VAL,
NIC, their physical mixture, and co-amorphous
mixtures were recorded using the Netzsch STA 409 C/CD instrument.
The samples were placed in sealed, non-hermetic aluminum pans and
scanned at a heating rate of 10.0 K/min under an argon atmosphere. 

Scanning Electron Microscopy A VEGA3
TESCAN scanning electron microscope was used to collect photo-micrographs
of co-amorphous mixtures and their components. The samples were mounted
on a metal stub with adhesive tape and observed without coating under
high vacuum. For each sample, 200× magnification was applied. 

Solution-State NMR Studies The 1 H NMR spectra were collected for co-amorphous formulations
in order to investigate contents of ethanol in the obtained formulations.
The 1 H NMR spectra were measured on a Bruker AV III 500
spectrometer in CDCl 3 with chemical shifts (δ) given
in ppm relative to TMS as an internal standard. 

Solubility Studies The solubility
of the co-amorphous dispersions obtained and free VAL and physical
mixture of VAL and NIC was determined in distilled water and phosphate
buffer (pH = 7.4) at 37 °C to simulate physiological conditions.
The relevant sample was weighted in order to obtain supersaturated
solution in water/phosphate buffer using a 20 mL test tube immersed
in a thermostated water bath (37 °C) and equipped with a magnetic
stirrer. The contents of the test tube were continuously stirred for
15 min; then, an additional amount of the relevant solvent (e.g.,
0.1 mL) was added to the tube until the test substance completely
dissolved, as determined with the macrocamera. The experiment was
performed in triplicate and solubility values were expressed as a
mean ± standard deviation. The solubility values for solvated
co-amorphous solid dispersions and physical mixture were recalculated
in reference to pure VAL, taking into account the content of VAL relative
to NIC and EtOH in the obtained solvated solid dispersions. 

Results and Discussion 3.1 Hydrogen-Bonding Interactions:
FT-IR Spectra,
DFT, QTAIM, and NCI Calculations The experimental FT-IR spectra
of VAL, NIC, their physical mixtures, and co-amorphous formulations
are presented in . The FT-IR spectrum of VAL shows two characteristic sharp peaks
at 1732 and 1602 cm –1 , corresponding to the υ(C=O) acid and υ(C=O) amide vibrations, respectively.
Broad, merged peaks with maxima at 3121 and 3420 cm –1 can be assigned to the υ(OH) and υ(NH) vibrations of
VAL, respectively. The FT-IR spectrum of NIC indicates the presence
of five characteristic absorption bands at 3367, 3160, 1683 cm –1 (with a shoulder at 1700 cm –1 ),
1593 , and 1575 cm –1 corresponding to the υ(NH 2 ) as , υ(NH 2 ) s , υ(C=O),
and two δ(NH) vibrations, respectively. Two narrow sharp peaks
at 1340 and 1203 cm –1 corresponding to the υ(C–N)
and υ(C–NH 2 ) vibrations are also observed
along with the ρ w (NH 2 ) broad peak at 778
cm –1 . The characteristic vibrations and band shifting
of the most important groups that can form hydrogen bonds are collected
in . The spectrum
of the VAL/NIC physical mixture reveals non-shifted peaks of single
components, in particular carbonyl peaks of VAL (1732 and 1602 cm –1 ) and NIC (1684 cm –1 with a shoulder
at 1700 cm –1 ), which has low intensity. A comparison
of the spectra indicates that major bands of the starting compounds
are shifted in the solid dispersions obtained. In case of the VAL/NIC
ball mill sample, three carbonyl peaks are significantly shifted:
υ(C=O) amide and υ(C=O) acid peaks of VAL and υ(C=O) peak of NIC are shifted from
1602, 1732 — , and 1683 to 1622, 1716 — , and 1675 cm –1 respectively. Two carbonyl peaks
are also significantly shifted in the case of VAL/NIC slurry: the
υ(C=O) amide peak of VAL is shifted from 1602
to 1649 cm –1 , whereas a υ(C=O) peak
of NIC is moved from 1683 to 1720 cm –1 . Smaller bands
shifts are observed in the case of the VAL/NIC LAG
solid dispersions where the υ(C=O) amide peak
of VAL is slightly shifted from 1602 to 1605 cm –1 , υ(C=O) peak of NIC is slightly moved from 1683 to
1681 cm –1 , and υ(C=O) acid peak of
VAL is shifted from 1732 to 1717 cm –1 . It is worth
noting that the υ(C=O) acid peak of VAL is
noticeably shifted only in the spectra of the VAL/NIC LAG and VAL/NIC
ball mill solid dispersions, suggesting that the interactions between
components in these formulations are different from those for the
abovementioned VAL/NIC slurry dispersion. However, the additional
peak at 1682 cm –1 corresponding to the unreacted
NIC is additionally visible in the spectrum of the VAL/NIC LAG 30
min dispersion. Moreover, carbonyl bands are merged and broadened
in the case of all formulations investigated, which suggests the formation
of supramolecular heterosynthons that involve interactions of these
C=O groups. The assignment and shift interpretation of the
υ(OH) and υ(NH) vibrations of VAL is difficult due to
peak broadening caused by the presence of ethanol OH groups (solvated
dispersions). In the case of the VAL/NIC slurry dispersion, υ(NH 2 ) as of NIC is shifted from 3367 to 3204 cm –1 and the υ(NH 2 ) s vibration
is shifted from 3160 to 3056 cm –1 . Smaller shifts
are observed in the case of the VAL/NIC ball mill solid dispersion,
where υ(NH 2 ) as of NIC is shifted from
3367 to 3352 cm –1 and the υ(NH 2 ) s vibration is shifted from 3160 to 3192 cm –1 . Such shifts in the υ(NH 2 ) frequencies are not
observed in the case of the VAL/NIC LAG dispersions, for which spectra
reveal only υ(NH 2 ) s vibration shifted
from 3160 to 3183 cm –1 (VAL/NIC LAG 30 min) and
3182 cm –1 (VAL/NIC LAG 60 min). Taking into account
the presence of an additional peak at 1682 cm –1 with
a shoulder at 1697 cm –1 in the FT-IR spectrum of
the VAL/NIC LAG 30 min dispersion, which corresponds to the υ(C=O)
peak of NIC and the υ(C=O) acid peak of VAL
at 1732 cm –1 , it can be assumed that the VAL/NIC
LAG 30 min dispersion is not fully co-amorphized and contains additionally
free VAL and NIC. In order to investigate what kind of intermolecular
interactions
could dominate in the synthesized co-amorphous solid dispersions,
two systems differing in the existing supramolecular heterosynthons
were designed and the theoretical IR spectra were calculated with
the hybrid functional B3LYP combined with the 6-311++G(d,p) basis
set . Then,
the experimental IR frequencies of synthesized co-amorphous solid
dispersions were compared with the calculated ones for VAL/NIC1 and
VAL/NIC2 .
In the designed VAL/NIC1 system, two hydrogen bonds, that is, N–H···O
and O–H···O hydrogen bonds, connect VAL and
NIC molecules into an acid–amide heterosynthon, which can be
described by the R 2 2(8) graph-set notation, whereas in the VAL/NIC2 system, a combination
of two hydrogen bonds (i.e., the first one between the tetrazolyl
NH group of VAL and amide oxygen of NIC and the second between the
amide oxygen of VAL and amide NH group of NIC) leads to the formation
of a ring with the descriptor R 2 2 (17). A detailed comparison of experimental vibrations of co-amorphous
solid dispersions with calculated frequencies for VAL/NIC1 and VAL/NIC2
is presented in the Supporting Information (Tables S7–S9). The comparative analysis of the experimental
and theoretical FT-IR
spectra shows that the interactions occurring in the VAL/NIC slurry
co-amorphous solid dispersion are mainly of the VAL/NIC1 type. A significant
shift to a lower wavenumber of the υ(NH 2 ) s vibration is observed both in the experimental VAL/NIC slurry and
calculated spectra. Moreover, the position and intensity of υ(C=O)
vibrations in the VAL/NIC slurry spectrum are analogous to those calculated
for VAL/NIC1 (two merged peaks for three υ(C=O) vibrations).
Smaller shifts, due to υ(NH 2 ) s and υ(NH 2 ) as vibrations, are observed in the case of the
calculated VAL/NIC2 system. The position and intensity of υ(C=O)
vibrations in VAL/NIC2 are different from in the case of VAL/NIC1.
This υ(C=O) vibration pattern, where three separate υ(C=O)
bands are observed, is analogous to VAL/NIC LAG 60 min and VAL/NIC
ball mill spectra; however, due to the overlapping of υ(C=O)
vibrations, the presence of coexisting VAL/NIC1-type interactions
in these samples may be suggested. The results of QTAIM analysis,
performed for two VAL/NIC heterodimers
at the ground state in the gas phase, showed the BCPs expected for
the interactions between the VAL and NIC molecules ( a). Further analysis of the
topological parameters listed in indicates that the N3–H3a···O3
interaction (BCP2 in VAL/NIC1) and N1–H1···O4
and N3–H3a···O3 interactions (BCP3 and BCP4,
respectively, in VAL/NIC2) are characterized by small values of electron
density and its Laplacian (0.023 < ρ BCP < 0.032,
0.094 < ∇ 2 ρ BCP < 0.123) and
fulfill the first three KP criteria for the existence of a hydrogen
bond. Taking into account the fact that the fourth criterion (i.e.,
Δ r H + Δ r A > 0 and Δ r H –
Δ r A > 0) is also met, all these
interactions may
be regarded as the hydrogen bonds. The positive values of ∇ 2 ρ BCP and H BCP values indicate that accordingly, these
three interactions may be classified as weak hydrogen bonds of electrostatic
nature. The fourth KP criterion is also fulfilled for the O2–H2···O4
interaction (BCP1) in the VAL/NIC1 heterodimer. The BCP for this interaction
is characterized by the highest positive values of charge density
and its Laplacian (ρ BCP = 0.0514 a.u, ∇ 2 ρ BCP = 0.1413 a.u), which both lie above
the upper limits of these properties proposed by KP (i.e., ρ BCP = 0.034 a.u, ∇ 2 ρ BCP =
0.139 a.u). The positive ∇ 2 ρ BCP value and negative H BCP value allow us to classify this interaction as a medium
H-bond with a partially covalent character.
The medium strength of the O2–H2···O4 hydrogen
bond is additionally confirmed by the value of hydrogen bond energy E bin = −15.68 kcal/mol. The isosurface
shape and color indicate that it is an interaction of type I, that
is, strong stabilizing interaction. Although other
hydrogen bonds between VAL and NIC molecules in
the considered heterodimers are also the interactions of this type,
the highest intensity of blue color of the isosurface and the lowest
sign(λ 2 )·ρ value in the case of the O2–H2···O4
hydrogen bond in the VAL/NIC1 heterosynthon are further confirmation
that it is the strongest interaction. In addition, the second hydrogen
bond forming this heterosynthon, that is, the N3–H3a···O3
hydrogen bond, is slightly stronger than the N3–H3a···O1
hydrogen bond in the VAL/NIC2 heterodimer. Hence, it can be assumed
that formation of the VAL/NIC1-type heterodimers is more preferred.
This assumption is in agreement with the experimental FT-IR results,
which suggest that VAL/NIC1-type interactions occur in the VAL/NIC
slurry co-amorphous solid dispersion. It is not so clear in the case
of the VAL/NIC LAG and ball mill solid dispersions where there are
probably heterodimers of both types. It can be suspected that VAL/NIC1
and VAL/NIC2 heterodimers do not form a long-range order, remaining
in a homogenous co-amorphous phase with a short-range order. This
is due to the fact that the formation of non-covalent interactions
connecting heterodimers with each other can be limited. For example,
the VAL molecule in VAL/NIC1 adopts a conformation, such that the
tetrazole ring is at an angle of 21.21° to the ring to which
it is attached. In this way, the donor tetrazolyl N1–H1 group
is isolated from any possible intermolecular hydrogen bonds which
could lead to the formation of a co-crystal . It is worth adding that the dihedral angle between
the planes of abovementioned two rings of the VAL molecule in the
crystal structure KIPLIG and KIPLEC is 57.51(1) and 48.37(5)°,
respectively. The N1–H1 bond in VAL/NIC1 is directed, so that
it participates in the formation of intramolecular contacts. They
are revealed in the form of a relatively large green isosurface on
the 3D NCI-plot ( b), which correspond to the sign (λ 2 )·ρ
values of c.a. −0.005 a.u. in the 2D NCI-plot. As our solid
dispersions are ethanol-solvated, we investigate the possible intermolecular
interactions of ethanol with the VAL/NIC heterodimer by designing
the (VAL/NIC1)EtOH system and doing the calculations in the framework
of QTAIM and NCI methods—as in the case of earlier structures.
The same, additional calculations were made for the VAL ethanol solvate
crystal (CSD refcode KIPLEC) to compare the energy of interactions
with ethanol. It can be seen that in the case of the VAL/NIC1 heterodimer,
ethanol can interact only with VAL’s tetrazole ring because
VAL’s carboxylic acid group is occupied by creating two strong
hydrogen bonds (one with a partially covalent character) with NIC
(binding energy −15.68 and −6.40 kcal/mol). The same
O5–H5···N2′ intermolecular interaction,
where the tetrazole ring acts as a hydrogen bond acceptor, occurs
in the VAL ethanol solvate crystal (VAL/VAL3). The calculated energy
of this interaction is −4.85 kcal/mol for the VAL/VAL3 and
−3.72 kcal/mol for the (VAL/NIC)EtOH. Therefore, it has been
shown that ethanol can form intermolecular interactions with the VAL/NIC
system, which were further confirmed by the fact that this solvent
could not be evaporated from the samples despite the use of high vacuum,
which suggests strong binding of the solvent. It is worth mentioning
that the previously described dihedral angle regarding the tetrazole
ring in (VAL/NIC1)EtOH is 41.50° (almost two times higher than
in VAL/NIC1 without ethanol); therefore, this interaction caused important
conformational changes in the VAL molecule. In order to compare
the energies of intermolecular hydrogen bonds
in VAL/NIC heterodimers with those identified in the crystal structures
of VAL and NIC, analogous QTAIM and NCI
calculations were conducted for molecular pairs cut out from these
crystals. VAL crystallizes in an orthorhombic space group P 2 1 2 1 2 1 in two possible
polymorphic forms E and F, which due to the presence of an ethanol
molecule in one of them differ in both the molecular conformation
and the packing motifs. In the crystal
structure of form E, the VAL molecules are connected by the O–H (carboxylic) ···N (tetrazole) (VAL/VAL1)
and N–H (tetrazole) ···O=C (carbonyl) (VAL/VAL2) hydrogen bonds into the two-dimensional
framework. In the crystal structure of the solvated form F (VAL/VAL3),
there are three different hydrogen bonds: one—between N–H
atoms of the tetrazole ring and carbonyl O atom, linking the VAL molecules
(i.e., N–H (tetrazole) ···O (carbonyl) ) and two bonds connecting the VAL molecule with the ethanol molecule
(i.e., O–H (carboxylic) ···O (ethanol) and O–H (ethanol) ···N (tetrazole) ). The obtained results show that the energy of intermolecular hydrogen
bonds in the NIC/NIC trimer is significantly lower than the energy
between VAL and NIC in the designed heterodimers . Based on the energy of intermolecular
interactions, one can conclude that the VAL/NIC1 heterodimer forms
stronger hydrogen bonds than VAL/VAL homodimers. Therefore, the abovementioned
data further confirm the preference of VAL to interact with NIC and
this way to form heterodimers instead of individual homodimers/trimers,
especially in the VAL/NIC1 system, where the intermolecular bonds
are the strongest of all systems studied. Due to the fact that
the solid dispersions were obtained in a solvated
form, additional DFT, QTAIM, and NCI calculations were carried out
for heterodimers optimized in ethanol. In general, the results obtained
both in the gas phase and in ethanol were comparable. The heterodimers
seemed to be more stable in the presence of ethanol due to a slightly
lower total energy of the system ( Tables S4a,b and S5a,b ). It was noted that the strongest intermolecular
hydrogen bonds (O2–H2···O4 in VAL/NIC1 and N1–H1···O4
in VAL/NIC2) were further strengthened in the presence of ethanol.
It is revealed by the highest binding energy of these hydrogen bonds
( Table S10 , SM) and elongation of the D(donor)–H
length and shortening of the H···A(acceptor) and D···A
lengths in the presence of ethanol ( Table S11 ). The interactions between solutes represented by the designed
heterodimers
and solvents (water and ethanol) were investigated by the PCM, first
described in 1981 by Tomasi et al. − Interactions with water are important to get the initial view of
the expected solubility of the designed heterodimers. Since the obtained
solid dispersions are ethanol-solvated, the solvation energies were
also calculated in relation to this solvent. Calculated free energies
of solvation, its components, and dipole moments for both heterodimers
are presented in . In general, the greater the value of solvation free energy is,
the higher the solubility is expected. As the
results of these studies, the VAL/NIC2 heterodimer was slightly
more soluble both in water and ethanol. Therefore, these heterodimers
may occur in solid dispersions, which contain more ethanol and exhibit
higher water solubility. The contributions of electrostatic and non-electrostatic
interactions to the solvation free energy are comparable in two heterodimers;
however, a slight predominance of non-electrostatic interactions was
observed in the case of VAL/NIC2. It has been reported that non-electrostatic
interactions, such as van der Waals interactions, play a major role
in solubilizing the pharmaceuticals, which
further confirms the assumption that the VAL/NIC2 heterodimer may
have a greater solubility than the VAL/NIC1. Moreover, in the case
of the VAL/NIC2 heterodimer, the difference in dipole moment values
in the gas phase and ethanol has been observed. The dipole moments
calculated in the solvent (both in water and ethanol) are higher than
those calculated in the gas phase—therefore, this type of heterodimers
should be more polar and more soluble in water (especially in the
presence of ethanol). This difference in dipole moments is not observed
in the case of the VAL/NIC1 heterodimer and (VAL/NIC1)EtOH. In case
of the (VAL/NIC1)EtOH system, values of free energy of solvation and
its cavitation component are much higher than the same values for
VAL/NIC1. Moreover, it can be seen that the presence of ethanol increases
the role of non-electrostatic interactions, which are an important
factor affecting solubility. Taking into account these results, it
can be suspected that VAL/NIC2-type heterodimers may dominate in the
VAL/NIC ball mill co-amorphous solid dispersion, causing higher solubility
of this sample, what has, in fact, been observed during the solubility
tests ( vide infra ). 3.2 Solid-State
NMR There is a lack of
data regarding the ssNMR studies of co-amorphous mixtures of VAL.
Although NIC is a commonly used stabilizer of amorphous drugs, the
number of ssNMR studies is mainly limited to its co-crystals. − It is worth noting that to obtain a high-quality 13 C
CP-MAS NMR spectrum of NIC, the RDs during the acquisition should
be extended to 300 s For this purpose, 13 C CP-MAS NMR spectra of components and co-amorphous formulations
were recorded with a spinning rate of 12 kHz and recycle delays of
both 5 and 300 s . The spectra recorded for VAL with RDs equal to 5 and 300
s were identical ( Figure S5 ), while it
was impossible to record the ssNMR spectrum of NIC with the short
RD = 5 s (due to very long relaxation times for this compound). However,
the 13 C signals of the latter were visible in the spectra
of co-amorphous mixtures and could be recorded at RD = 5 s. This may
be due to the fact that relaxation times for new formulations were
shortened in comparison for the starting components, which confirms
formation of a new phase. Similar results have been reported for the
NIC co-crystals. − Moreover, the shifted, broadened, and merged carbonyl
peaks in co-amorphous formulation spectra (δ = 160–180
ppm) indicate the formation of non-covalent bonds that involve the
C=O groups. It is especially evident in the case of fully co-amorphous
VAL/NIC slurry and VAL/NIC ball mill samples. In the case of the VAL/NIC
LAG 30 min sample, the ssNMR spectrum, recorded with RD = 300 s, revealed
strong peaks, which correspond to the signals of NIC, so it can be
assumed that this sample contains additionally unreacted NIC (which
was also confirmed by other analyses). 3.3 Differential Scanning Calorimetry The DSC thermograms of the VAL/NIC co-amorphous mixtures and the
VAL/NIC physical mixture along with the insert of DSC thermograms
of components (VAL and NIC) are presented in . The NIC spectrum showed a single melting
endothermic peak at 135.3 °C, whereas the VAL revealed the melting
endothermic peak ( T m ) with the maxima
at 117.9 °C ( , insert). The physical mixture of VAL and NIC showed a single
broad endothermic peak at 116.0 °C and two glass transition events
( T g ) corresponding to NIC (45.5 °C)
and VAL (65.6 °C). What is important is that the peaks corresponding to the VAL/NIC
physical mixture and pure components disappeared in thermograms of
the co-amorphous dispersions obtained. However, new melting points
do not appear, which indicates that the formed samples are amorphous.
The co-amorphous formulations studied exhibited two endothermic events:
first, glass transition at 44.0 °C (VAL/NIC slurry), 46.1 °C
(VAL/NIC LAG 30 min), 45.3 °C (VAL/NIC LAG 60 min), and 42.3
°C (VAL/NIC ball mill) and second, vaporization of ethanol ( T evap (EtOH)) at 94.7 °C (VAL/NIC slurry),
85.5 °C (VAL/NIC LAG 30 min), 92.6 °C (VAL/NIC LAG 60 min),
and 89.7 °C (VAL/NIC ball mill). All studied co-amorphous mixtures exhibited a single glass transition
event, which indicated that they are homogeneous in contrast to their
physical mixture, which exhibited two separate glass transition events. In case of the VAL/NIC LAG 30 min, a small peak
with a maximum at 133.1 °C corresponding to the melting peak
of NIC (135.3 °C) is visible, which again confirms the presence
of additional unreacted NIC in a sample. This analysis clearly indicates
that elongation of the grinding time from 30 min (VAL/NIC LAG 30 min)
to 60 min (VAL/NIC LAG 60 min) increases the conversion of components
to give co-amorphous dispersion. 3.4 Solution-State
NMR Studies Because
the vaporization peak from ethanol was found in DSC thermograms of
all co-amorphous dispersions , it could be expected that the synthesized formulations
were obtained in the form of ethanol solvates. In order to quantitatively
estimate the amount of ethanol, the solution-state 1 H NMR
spectra were recorded ( Figure S9 ). The
ethanol content was sensitive to the degree of amorphization. The
calculated amounts of ethanol in fully amorphized dispersions VAL/NIC
slurry and VAL/NIC ball mill and in the nearly amorphized VAL/NIC
LAG 60 min sample remained in the range of 4.43–6.79% (by moles),
while in the less-amorphized VAL/NIC LAG 30 min sample, the ethanol
content dropped to 1.47% (by moles). 3.5 X-ray
Powder Diffraction The XRPD
patterns of co-amorphous solid dispersions (VAL/NIC LAG 30 min, VAL/NIC
LAG 60 min, VAL/NIC slurry, and VAL/NIC ball mill), their physical
mixtures in a 1:1 molar and weight ratios, and their components (VAL
and NIC) are presented in . Detailed analysis of the XRPD pattern of VAL is given
in the Supporting Information (Figure S7).
The XRPD pattern of the NIC sample used in this study can be accurately
matched to the simulated XRPD pattern, which was generated from the
deposited X-ray data file at the Cambridge Structure Database (CSD—refcode
NICOAM06) using Mercury software ( Figure S8 ). The XRPD analysis revealed that the pattern of the co-amorphous
mixtures obtained is different from that for the VAL/NIC physical
mixtures. Characteristic reflections of components (VAL and NIC) completely
disappeared in the XRPD patterns of the VAL/NIC slurry and VAL/NIC
ball mill. The appearance of an amorphous halo in the XRPD pattern
of the solid dispersions demonstrated the amorphous nature of the
obtained drug dispersions. It can be also concluded that the VAL/NIC
slurry and VAL/NIC ball mill dispersions are different from the VAL/NIC
LAG (30 and 60 min) dispersions, which exhibit additional three small
reflections at 2θ: 14.88, 25.53, and 27.15°. These reflections
correspond to the most intensive reflections in the XRPD pattern generated
based on the crystal structure of NICOAM06. Thus, they can be related
to incomplete amorphization of NIC in VAL/NIC LAG solid dispersions.
Moreover, it can be estimated that the amount of non-amorphized NIC
is greater in the VAL/NIC LAG 30 min than in the VAL/NIC LAG 60 min
solid dispersion, which suggests that longer grinding time results
in a more amorphous product; however, the grinding time of 60 min
is still too short to fully co-amorphize this system with this technique.
All of the abovementioned data prove that as a result of intermolecular
interactions, the synthesized VAL solid dispersions formed new phases.
The stability of these co-amorphous products, and thus their resistance
to recrystallization, was tested under 25 °C, 0% RH conditions
. The fact
of appearance of a crystalline phase of any of the components of the
co-amorphous product (VAL or NIC) indicates a loss of stability. Of these two components, NIC has much greater
tendency to crystallization than VAL. Therefore, it could be expected
that during the loss of amorphous stability, the reflections from
NIC would first reveal on the diffraction pattern of the product.
In fact, the XRPD method showed the presence of at least one reflection
derived from the NIC crystalline phase present in the recrystallized
products. In case of the VAL/NIC ball mill sample, first small reflection
has been observed after 11 months of storage what confirms the excellent
durability of this co-amorphous sample. Recrystallization of the VAL/NIC
slurry sample was observed after 9 months of storage. 3.6 Scanning Electron Microscopy The
SEM image overview of pure VAL, NIC, and co-amorphous formulations
is presented in . The SEM results reveal that VAL occurs in the form of irregular
particles (up to 200 μm) with a rough surface and NIC is in
the form of spherical rough grains (50–100 μm). The obtained
particles of co-amorphous mixtures exhibited irregular shapes, different
from those recorded for the pure VAL and NIC. The size of the VAL/NIC
LAG 60 min particles is <50 μm, and the size of the VAL/NIC
LAG 30 min particles remains in the range of 50–200 μm,
whereas the VAL/NIC slurry particles are larger (>200 μm).
The
VAL/NIC ball mill particles remain in the range of 50–200 μm
and form thin solid plates. 3.7 Solubility
Studies The main goal
of the solid dispersion synthesis is improving solubility of poorly
soluble drugs (in this case—VAL). The solubility studies are
performed in suitable medium simulating physiological conditions.
In this paper, the solubility of the free VAL and four co-amorphous
mixtures was investigated in the phosphate buffer at the physiological
pH 7.4 and in distilled water at 37 °C . The results showed that the VAL/NIC LAG
30 min and VAL/NIC LAG 60 min solid dispersions exhibited similar
solubility both in the phosphate buffer and water. These co-amorphous
mixtures caused a 3.5-fold increase in solubility of VAL in water
and only a 1.2-fold increase in solubility in the phosphate buffer.
On the other hand, co-amorphous VAL/NIC slurry solid dispersion slightly
increased solubility of VAL in the phosphate buffer, whereas a significant
14-fold increase in solubility was observed in water. An enhanced
24-fold higher water solubility than free VAL showed in the VAL/NIC
ball mill solid dispersion. Therefore, taking into account the difference
in the solubility of the VAL/NIC LAG (30 and 60 min) dispersions and
the VAL/NIC slurry and VAL/NIC ball mill dispersions, it can be assumed
that these co-amorphous formulations involve different intermolecular
interactions with different morphologies of the powders, which was
confirmed here by the relevant analyses and calculations. Interestingly,
the solubility of the VAL/NIC ball mill sample with first signs of
recrystallization remained the same in phosphate buffer, whereas its
solubility in water decreased. This further confirms that co-amorphization
significantly improves the solubility in water and has lesser effect
on the solubility in the buffer. This also supports the thesis that
lower solubility of VAL/NIC LAG samples in water is a consequence
of their not fully amorphous nature. 

Hydrogen-Bonding Interactions:
FT-IR Spectra,
DFT, QTAIM, and NCI Calculations The experimental FT-IR spectra
of VAL, NIC, their physical mixtures, and co-amorphous formulations
are presented in . The FT-IR spectrum of VAL shows two characteristic sharp peaks
at 1732 and 1602 cm –1 , corresponding to the υ(C=O) acid and υ(C=O) amide vibrations, respectively.
Broad, merged peaks with maxima at 3121 and 3420 cm –1 can be assigned to the υ(OH) and υ(NH) vibrations of
VAL, respectively. The FT-IR spectrum of NIC indicates the presence
of five characteristic absorption bands at 3367, 3160, 1683 cm –1 (with a shoulder at 1700 cm –1 ),
1593 , and 1575 cm –1 corresponding to the υ(NH 2 ) as , υ(NH 2 ) s , υ(C=O),
and two δ(NH) vibrations, respectively. Two narrow sharp peaks
at 1340 and 1203 cm –1 corresponding to the υ(C–N)
and υ(C–NH 2 ) vibrations are also observed
along with the ρ w (NH 2 ) broad peak at 778
cm –1 . The characteristic vibrations and band shifting
of the most important groups that can form hydrogen bonds are collected
in . The spectrum
of the VAL/NIC physical mixture reveals non-shifted peaks of single
components, in particular carbonyl peaks of VAL (1732 and 1602 cm –1 ) and NIC (1684 cm –1 with a shoulder
at 1700 cm –1 ), which has low intensity. A comparison
of the spectra indicates that major bands of the starting compounds
are shifted in the solid dispersions obtained. In case of the VAL/NIC
ball mill sample, three carbonyl peaks are significantly shifted:
υ(C=O) amide and υ(C=O) acid peaks of VAL and υ(C=O) peak of NIC are shifted from
1602, 1732 — , and 1683 to 1622, 1716 — , and 1675 cm –1 respectively. Two carbonyl peaks
are also significantly shifted in the case of VAL/NIC slurry: the
υ(C=O) amide peak of VAL is shifted from 1602
to 1649 cm –1 , whereas a υ(C=O) peak
of NIC is moved from 1683 to 1720 cm –1 . Smaller bands
shifts are observed in the case of the VAL/NIC LAG
solid dispersions where the υ(C=O) amide peak
of VAL is slightly shifted from 1602 to 1605 cm –1 , υ(C=O) peak of NIC is slightly moved from 1683 to
1681 cm –1 , and υ(C=O) acid peak of
VAL is shifted from 1732 to 1717 cm –1 . It is worth
noting that the υ(C=O) acid peak of VAL is
noticeably shifted only in the spectra of the VAL/NIC LAG and VAL/NIC
ball mill solid dispersions, suggesting that the interactions between
components in these formulations are different from those for the
abovementioned VAL/NIC slurry dispersion. However, the additional
peak at 1682 cm –1 corresponding to the unreacted
NIC is additionally visible in the spectrum of the VAL/NIC LAG 30
min dispersion. Moreover, carbonyl bands are merged and broadened
in the case of all formulations investigated, which suggests the formation
of supramolecular heterosynthons that involve interactions of these
C=O groups. The assignment and shift interpretation of the
υ(OH) and υ(NH) vibrations of VAL is difficult due to
peak broadening caused by the presence of ethanol OH groups (solvated
dispersions). In the case of the VAL/NIC slurry dispersion, υ(NH 2 ) as of NIC is shifted from 3367 to 3204 cm –1 and the υ(NH 2 ) s vibration
is shifted from 3160 to 3056 cm –1 . Smaller shifts
are observed in the case of the VAL/NIC ball mill solid dispersion,
where υ(NH 2 ) as of NIC is shifted from
3367 to 3352 cm –1 and the υ(NH 2 ) s vibration is shifted from 3160 to 3192 cm –1 . Such shifts in the υ(NH 2 ) frequencies are not
observed in the case of the VAL/NIC LAG dispersions, for which spectra
reveal only υ(NH 2 ) s vibration shifted
from 3160 to 3183 cm –1 (VAL/NIC LAG 30 min) and
3182 cm –1 (VAL/NIC LAG 60 min). Taking into account
the presence of an additional peak at 1682 cm –1 with
a shoulder at 1697 cm –1 in the FT-IR spectrum of
the VAL/NIC LAG 30 min dispersion, which corresponds to the υ(C=O)
peak of NIC and the υ(C=O) acid peak of VAL
at 1732 cm –1 , it can be assumed that the VAL/NIC
LAG 30 min dispersion is not fully co-amorphized and contains additionally
free VAL and NIC. In order to investigate what kind of intermolecular
interactions
could dominate in the synthesized co-amorphous solid dispersions,
two systems differing in the existing supramolecular heterosynthons
were designed and the theoretical IR spectra were calculated with
the hybrid functional B3LYP combined with the 6-311++G(d,p) basis
set . Then,
the experimental IR frequencies of synthesized co-amorphous solid
dispersions were compared with the calculated ones for VAL/NIC1 and
VAL/NIC2 .
In the designed VAL/NIC1 system, two hydrogen bonds, that is, N–H···O
and O–H···O hydrogen bonds, connect VAL and
NIC molecules into an acid–amide heterosynthon, which can be
described by the R 2 2(8) graph-set notation, whereas in the VAL/NIC2 system, a combination
of two hydrogen bonds (i.e., the first one between the tetrazolyl
NH group of VAL and amide oxygen of NIC and the second between the
amide oxygen of VAL and amide NH group of NIC) leads to the formation
of a ring with the descriptor R 2 2 (17). A detailed comparison of experimental vibrations of co-amorphous
solid dispersions with calculated frequencies for VAL/NIC1 and VAL/NIC2
is presented in the Supporting Information (Tables S7–S9). The comparative analysis of the experimental
and theoretical FT-IR
spectra shows that the interactions occurring in the VAL/NIC slurry
co-amorphous solid dispersion are mainly of the VAL/NIC1 type. A significant
shift to a lower wavenumber of the υ(NH 2 ) s vibration is observed both in the experimental VAL/NIC slurry and
calculated spectra. Moreover, the position and intensity of υ(C=O)
vibrations in the VAL/NIC slurry spectrum are analogous to those calculated
for VAL/NIC1 (two merged peaks for three υ(C=O) vibrations).
Smaller shifts, due to υ(NH 2 ) s and υ(NH 2 ) as vibrations, are observed in the case of the
calculated VAL/NIC2 system. The position and intensity of υ(C=O)
vibrations in VAL/NIC2 are different from in the case of VAL/NIC1.
This υ(C=O) vibration pattern, where three separate υ(C=O)
bands are observed, is analogous to VAL/NIC LAG 60 min and VAL/NIC
ball mill spectra; however, due to the overlapping of υ(C=O)
vibrations, the presence of coexisting VAL/NIC1-type interactions
in these samples may be suggested. The results of QTAIM analysis,
performed for two VAL/NIC heterodimers
at the ground state in the gas phase, showed the BCPs expected for
the interactions between the VAL and NIC molecules ( a). Further analysis of the
topological parameters listed in indicates that the N3–H3a···O3
interaction (BCP2 in VAL/NIC1) and N1–H1···O4
and N3–H3a···O3 interactions (BCP3 and BCP4,
respectively, in VAL/NIC2) are characterized by small values of electron
density and its Laplacian (0.023 < ρ BCP < 0.032,
0.094 < ∇ 2 ρ BCP < 0.123) and
fulfill the first three KP criteria for the existence of a hydrogen
bond. Taking into account the fact that the fourth criterion (i.e.,
Δ r H + Δ r A > 0 and Δ r H –
Δ r A > 0) is also met, all these
interactions may
be regarded as the hydrogen bonds. The positive values of ∇ 2 ρ BCP and H BCP values indicate that accordingly, these
three interactions may be classified as weak hydrogen bonds of electrostatic
nature. The fourth KP criterion is also fulfilled for the O2–H2···O4
interaction (BCP1) in the VAL/NIC1 heterodimer. The BCP for this interaction
is characterized by the highest positive values of charge density
and its Laplacian (ρ BCP = 0.0514 a.u, ∇ 2 ρ BCP = 0.1413 a.u), which both lie above
the upper limits of these properties proposed by KP (i.e., ρ BCP = 0.034 a.u, ∇ 2 ρ BCP =
0.139 a.u). The positive ∇ 2 ρ BCP value and negative H BCP value allow us to classify this interaction as a medium
H-bond with a partially covalent character.
The medium strength of the O2–H2···O4 hydrogen
bond is additionally confirmed by the value of hydrogen bond energy E bin = −15.68 kcal/mol. The isosurface
shape and color indicate that it is an interaction of type I, that
is, strong stabilizing interaction. Although other
hydrogen bonds between VAL and NIC molecules in
the considered heterodimers are also the interactions of this type,
the highest intensity of blue color of the isosurface and the lowest
sign(λ 2 )·ρ value in the case of the O2–H2···O4
hydrogen bond in the VAL/NIC1 heterosynthon are further confirmation
that it is the strongest interaction. In addition, the second hydrogen
bond forming this heterosynthon, that is, the N3–H3a···O3
hydrogen bond, is slightly stronger than the N3–H3a···O1
hydrogen bond in the VAL/NIC2 heterodimer. Hence, it can be assumed
that formation of the VAL/NIC1-type heterodimers is more preferred.
This assumption is in agreement with the experimental FT-IR results,
which suggest that VAL/NIC1-type interactions occur in the VAL/NIC
slurry co-amorphous solid dispersion. It is not so clear in the case
of the VAL/NIC LAG and ball mill solid dispersions where there are
probably heterodimers of both types. It can be suspected that VAL/NIC1
and VAL/NIC2 heterodimers do not form a long-range order, remaining
in a homogenous co-amorphous phase with a short-range order. This
is due to the fact that the formation of non-covalent interactions
connecting heterodimers with each other can be limited. For example,
the VAL molecule in VAL/NIC1 adopts a conformation, such that the
tetrazole ring is at an angle of 21.21° to the ring to which
it is attached. In this way, the donor tetrazolyl N1–H1 group
is isolated from any possible intermolecular hydrogen bonds which
could lead to the formation of a co-crystal . It is worth adding that the dihedral angle between
the planes of abovementioned two rings of the VAL molecule in the
crystal structure KIPLIG and KIPLEC is 57.51(1) and 48.37(5)°,
respectively. The N1–H1 bond in VAL/NIC1 is directed, so that
it participates in the formation of intramolecular contacts. They
are revealed in the form of a relatively large green isosurface on
the 3D NCI-plot ( b), which correspond to the sign (λ 2 )·ρ
values of c.a. −0.005 a.u. in the 2D NCI-plot. As our solid
dispersions are ethanol-solvated, we investigate the possible intermolecular
interactions of ethanol with the VAL/NIC heterodimer by designing
the (VAL/NIC1)EtOH system and doing the calculations in the framework
of QTAIM and NCI methods—as in the case of earlier structures.
The same, additional calculations were made for the VAL ethanol solvate
crystal (CSD refcode KIPLEC) to compare the energy of interactions
with ethanol. It can be seen that in the case of the VAL/NIC1 heterodimer,
ethanol can interact only with VAL’s tetrazole ring because
VAL’s carboxylic acid group is occupied by creating two strong
hydrogen bonds (one with a partially covalent character) with NIC
(binding energy −15.68 and −6.40 kcal/mol). The same
O5–H5···N2′ intermolecular interaction,
where the tetrazole ring acts as a hydrogen bond acceptor, occurs
in the VAL ethanol solvate crystal (VAL/VAL3). The calculated energy
of this interaction is −4.85 kcal/mol for the VAL/VAL3 and
−3.72 kcal/mol for the (VAL/NIC)EtOH. Therefore, it has been
shown that ethanol can form intermolecular interactions with the VAL/NIC
system, which were further confirmed by the fact that this solvent
could not be evaporated from the samples despite the use of high vacuum,
which suggests strong binding of the solvent. It is worth mentioning
that the previously described dihedral angle regarding the tetrazole
ring in (VAL/NIC1)EtOH is 41.50° (almost two times higher than
in VAL/NIC1 without ethanol); therefore, this interaction caused important
conformational changes in the VAL molecule. In order to compare
the energies of intermolecular hydrogen bonds
in VAL/NIC heterodimers with those identified in the crystal structures
of VAL and NIC, analogous QTAIM and NCI
calculations were conducted for molecular pairs cut out from these
crystals. VAL crystallizes in an orthorhombic space group P 2 1 2 1 2 1 in two possible
polymorphic forms E and F, which due to the presence of an ethanol
molecule in one of them differ in both the molecular conformation
and the packing motifs. In the crystal
structure of form E, the VAL molecules are connected by the O–H (carboxylic) ···N (tetrazole) (VAL/VAL1)
and N–H (tetrazole) ···O=C (carbonyl) (VAL/VAL2) hydrogen bonds into the two-dimensional
framework. In the crystal structure of the solvated form F (VAL/VAL3),
there are three different hydrogen bonds: one—between N–H
atoms of the tetrazole ring and carbonyl O atom, linking the VAL molecules
(i.e., N–H (tetrazole) ···O (carbonyl) ) and two bonds connecting the VAL molecule with the ethanol molecule
(i.e., O–H (carboxylic) ···O (ethanol) and O–H (ethanol) ···N (tetrazole) ). The obtained results show that the energy of intermolecular hydrogen
bonds in the NIC/NIC trimer is significantly lower than the energy
between VAL and NIC in the designed heterodimers . Based on the energy of intermolecular
interactions, one can conclude that the VAL/NIC1 heterodimer forms
stronger hydrogen bonds than VAL/VAL homodimers. Therefore, the abovementioned
data further confirm the preference of VAL to interact with NIC and
this way to form heterodimers instead of individual homodimers/trimers,
especially in the VAL/NIC1 system, where the intermolecular bonds
are the strongest of all systems studied. Due to the fact that
the solid dispersions were obtained in a solvated
form, additional DFT, QTAIM, and NCI calculations were carried out
for heterodimers optimized in ethanol. In general, the results obtained
both in the gas phase and in ethanol were comparable. The heterodimers
seemed to be more stable in the presence of ethanol due to a slightly
lower total energy of the system ( Tables S4a,b and S5a,b ). It was noted that the strongest intermolecular
hydrogen bonds (O2–H2···O4 in VAL/NIC1 and N1–H1···O4
in VAL/NIC2) were further strengthened in the presence of ethanol.
It is revealed by the highest binding energy of these hydrogen bonds
( Table S10 , SM) and elongation of the D(donor)–H
length and shortening of the H···A(acceptor) and D···A
lengths in the presence of ethanol ( Table S11 ). The interactions between solutes represented by the designed
heterodimers
and solvents (water and ethanol) were investigated by the PCM, first
described in 1981 by Tomasi et al. − Interactions with water are important to get the initial view of
the expected solubility of the designed heterodimers. Since the obtained
solid dispersions are ethanol-solvated, the solvation energies were
also calculated in relation to this solvent. Calculated free energies
of solvation, its components, and dipole moments for both heterodimers
are presented in . In general, the greater the value of solvation free energy is,
the higher the solubility is expected. As the
results of these studies, the VAL/NIC2 heterodimer was slightly
more soluble both in water and ethanol. Therefore, these heterodimers
may occur in solid dispersions, which contain more ethanol and exhibit
higher water solubility. The contributions of electrostatic and non-electrostatic
interactions to the solvation free energy are comparable in two heterodimers;
however, a slight predominance of non-electrostatic interactions was
observed in the case of VAL/NIC2. It has been reported that non-electrostatic
interactions, such as van der Waals interactions, play a major role
in solubilizing the pharmaceuticals, which
further confirms the assumption that the VAL/NIC2 heterodimer may
have a greater solubility than the VAL/NIC1. Moreover, in the case
of the VAL/NIC2 heterodimer, the difference in dipole moment values
in the gas phase and ethanol has been observed. The dipole moments
calculated in the solvent (both in water and ethanol) are higher than
those calculated in the gas phase—therefore, this type of heterodimers
should be more polar and more soluble in water (especially in the
presence of ethanol). This difference in dipole moments is not observed
in the case of the VAL/NIC1 heterodimer and (VAL/NIC1)EtOH. In case
of the (VAL/NIC1)EtOH system, values of free energy of solvation and
its cavitation component are much higher than the same values for
VAL/NIC1. Moreover, it can be seen that the presence of ethanol increases
the role of non-electrostatic interactions, which are an important
factor affecting solubility. Taking into account these results, it
can be suspected that VAL/NIC2-type heterodimers may dominate in the
VAL/NIC ball mill co-amorphous solid dispersion, causing higher solubility
of this sample, what has, in fact, been observed during the solubility
tests ( vide infra ). 

Solid-State
NMR There is a lack of
data regarding the ssNMR studies of co-amorphous mixtures of VAL.
Although NIC is a commonly used stabilizer of amorphous drugs, the
number of ssNMR studies is mainly limited to its co-crystals. − It is worth noting that to obtain a high-quality 13 C
CP-MAS NMR spectrum of NIC, the RDs during the acquisition should
be extended to 300 s For this purpose, 13 C CP-MAS NMR spectra of components and co-amorphous formulations
were recorded with a spinning rate of 12 kHz and recycle delays of
both 5 and 300 s . The spectra recorded for VAL with RDs equal to 5 and 300
s were identical ( Figure S5 ), while it
was impossible to record the ssNMR spectrum of NIC with the short
RD = 5 s (due to very long relaxation times for this compound). However,
the 13 C signals of the latter were visible in the spectra
of co-amorphous mixtures and could be recorded at RD = 5 s. This may
be due to the fact that relaxation times for new formulations were
shortened in comparison for the starting components, which confirms
formation of a new phase. Similar results have been reported for the
NIC co-crystals. − Moreover, the shifted, broadened, and merged carbonyl
peaks in co-amorphous formulation spectra (δ = 160–180
ppm) indicate the formation of non-covalent bonds that involve the
C=O groups. It is especially evident in the case of fully co-amorphous
VAL/NIC slurry and VAL/NIC ball mill samples. In the case of the VAL/NIC
LAG 30 min sample, the ssNMR spectrum, recorded with RD = 300 s, revealed
strong peaks, which correspond to the signals of NIC, so it can be
assumed that this sample contains additionally unreacted NIC (which
was also confirmed by other analyses). 

Differential Scanning Calorimetry The DSC thermograms of the VAL/NIC co-amorphous mixtures and the
VAL/NIC physical mixture along with the insert of DSC thermograms
of components (VAL and NIC) are presented in . The NIC spectrum showed a single melting
endothermic peak at 135.3 °C, whereas the VAL revealed the melting
endothermic peak ( T m ) with the maxima
at 117.9 °C ( , insert). The physical mixture of VAL and NIC showed a single
broad endothermic peak at 116.0 °C and two glass transition events
( T g ) corresponding to NIC (45.5 °C)
and VAL (65.6 °C). What is important is that the peaks corresponding to the VAL/NIC
physical mixture and pure components disappeared in thermograms of
the co-amorphous dispersions obtained. However, new melting points
do not appear, which indicates that the formed samples are amorphous.
The co-amorphous formulations studied exhibited two endothermic events:
first, glass transition at 44.0 °C (VAL/NIC slurry), 46.1 °C
(VAL/NIC LAG 30 min), 45.3 °C (VAL/NIC LAG 60 min), and 42.3
°C (VAL/NIC ball mill) and second, vaporization of ethanol ( T evap (EtOH)) at 94.7 °C (VAL/NIC slurry),
85.5 °C (VAL/NIC LAG 30 min), 92.6 °C (VAL/NIC LAG 60 min),
and 89.7 °C (VAL/NIC ball mill). All studied co-amorphous mixtures exhibited a single glass transition
event, which indicated that they are homogeneous in contrast to their
physical mixture, which exhibited two separate glass transition events. In case of the VAL/NIC LAG 30 min, a small peak
with a maximum at 133.1 °C corresponding to the melting peak
of NIC (135.3 °C) is visible, which again confirms the presence
of additional unreacted NIC in a sample. This analysis clearly indicates
that elongation of the grinding time from 30 min (VAL/NIC LAG 30 min)
to 60 min (VAL/NIC LAG 60 min) increases the conversion of components
to give co-amorphous dispersion. 

Solution-State
NMR Studies Because
the vaporization peak from ethanol was found in DSC thermograms of
all co-amorphous dispersions , it could be expected that the synthesized formulations
were obtained in the form of ethanol solvates. In order to quantitatively
estimate the amount of ethanol, the solution-state 1 H NMR
spectra were recorded ( Figure S9 ). The
ethanol content was sensitive to the degree of amorphization. The
calculated amounts of ethanol in fully amorphized dispersions VAL/NIC
slurry and VAL/NIC ball mill and in the nearly amorphized VAL/NIC
LAG 60 min sample remained in the range of 4.43–6.79% (by moles),
while in the less-amorphized VAL/NIC LAG 30 min sample, the ethanol
content dropped to 1.47% (by moles). 

X-ray
Powder Diffraction The XRPD
patterns of co-amorphous solid dispersions (VAL/NIC LAG 30 min, VAL/NIC
LAG 60 min, VAL/NIC slurry, and VAL/NIC ball mill), their physical
mixtures in a 1:1 molar and weight ratios, and their components (VAL
and NIC) are presented in . Detailed analysis of the XRPD pattern of VAL is given
in the Supporting Information (Figure S7).
The XRPD pattern of the NIC sample used in this study can be accurately
matched to the simulated XRPD pattern, which was generated from the
deposited X-ray data file at the Cambridge Structure Database (CSD—refcode
NICOAM06) using Mercury software ( Figure S8 ). The XRPD analysis revealed that the pattern of the co-amorphous
mixtures obtained is different from that for the VAL/NIC physical
mixtures. Characteristic reflections of components (VAL and NIC) completely
disappeared in the XRPD patterns of the VAL/NIC slurry and VAL/NIC
ball mill. The appearance of an amorphous halo in the XRPD pattern
of the solid dispersions demonstrated the amorphous nature of the
obtained drug dispersions. It can be also concluded that the VAL/NIC
slurry and VAL/NIC ball mill dispersions are different from the VAL/NIC
LAG (30 and 60 min) dispersions, which exhibit additional three small
reflections at 2θ: 14.88, 25.53, and 27.15°. These reflections
correspond to the most intensive reflections in the XRPD pattern generated
based on the crystal structure of NICOAM06. Thus, they can be related
to incomplete amorphization of NIC in VAL/NIC LAG solid dispersions.
Moreover, it can be estimated that the amount of non-amorphized NIC
is greater in the VAL/NIC LAG 30 min than in the VAL/NIC LAG 60 min
solid dispersion, which suggests that longer grinding time results
in a more amorphous product; however, the grinding time of 60 min
is still too short to fully co-amorphize this system with this technique.
All of the abovementioned data prove that as a result of intermolecular
interactions, the synthesized VAL solid dispersions formed new phases.
The stability of these co-amorphous products, and thus their resistance
to recrystallization, was tested under 25 °C, 0% RH conditions
. The fact
of appearance of a crystalline phase of any of the components of the
co-amorphous product (VAL or NIC) indicates a loss of stability. Of these two components, NIC has much greater
tendency to crystallization than VAL. Therefore, it could be expected
that during the loss of amorphous stability, the reflections from
NIC would first reveal on the diffraction pattern of the product.
In fact, the XRPD method showed the presence of at least one reflection
derived from the NIC crystalline phase present in the recrystallized
products. In case of the VAL/NIC ball mill sample, first small reflection
has been observed after 11 months of storage what confirms the excellent
durability of this co-amorphous sample. Recrystallization of the VAL/NIC
slurry sample was observed after 9 months of storage. 

Scanning Electron Microscopy The
SEM image overview of pure VAL, NIC, and co-amorphous formulations
is presented in . The SEM results reveal that VAL occurs in the form of irregular
particles (up to 200 μm) with a rough surface and NIC is in
the form of spherical rough grains (50–100 μm). The obtained
particles of co-amorphous mixtures exhibited irregular shapes, different
from those recorded for the pure VAL and NIC. The size of the VAL/NIC
LAG 60 min particles is <50 μm, and the size of the VAL/NIC
LAG 30 min particles remains in the range of 50–200 μm,
whereas the VAL/NIC slurry particles are larger (>200 μm).
The
VAL/NIC ball mill particles remain in the range of 50–200 μm
and form thin solid plates. 

Solubility
Studies The main goal
of the solid dispersion synthesis is improving solubility of poorly
soluble drugs (in this case—VAL). The solubility studies are
performed in suitable medium simulating physiological conditions.
In this paper, the solubility of the free VAL and four co-amorphous
mixtures was investigated in the phosphate buffer at the physiological
pH 7.4 and in distilled water at 37 °C . The results showed that the VAL/NIC LAG
30 min and VAL/NIC LAG 60 min solid dispersions exhibited similar
solubility both in the phosphate buffer and water. These co-amorphous
mixtures caused a 3.5-fold increase in solubility of VAL in water
and only a 1.2-fold increase in solubility in the phosphate buffer.
On the other hand, co-amorphous VAL/NIC slurry solid dispersion slightly
increased solubility of VAL in the phosphate buffer, whereas a significant
14-fold increase in solubility was observed in water. An enhanced
24-fold higher water solubility than free VAL showed in the VAL/NIC
ball mill solid dispersion. Therefore, taking into account the difference
in the solubility of the VAL/NIC LAG (30 and 60 min) dispersions and
the VAL/NIC slurry and VAL/NIC ball mill dispersions, it can be assumed
that these co-amorphous formulations involve different intermolecular
interactions with different morphologies of the powders, which was
confirmed here by the relevant analyses and calculations. Interestingly,
the solubility of the VAL/NIC ball mill sample with first signs of
recrystallization remained the same in phosphate buffer, whereas its
solubility in water decreased. This further confirms that co-amorphization
significantly improves the solubility in water and has lesser effect
on the solubility in the buffer. This also supports the thesis that
lower solubility of VAL/NIC LAG samples in water is a consequence
of their not fully amorphous nature. 

Conclusions Four solvated solid dispersions of the
hypertension drug VAL with
enhanced solubility in water and the phosphate buffer were synthesized.
NIC, as a nutraceutical, was used as a suitable co-former to give
dually acting pharmaceutical solid dispersions. The VAL/NIC slurry
and VAL/NIC ball mill dispersions revealed full amorphization and
the best solubility. These compositions could be used to treat two
seemingly unrelated diseases, such as hypertension and the COVID-19
disease. This is due to the fact that
SARS-CoV-2 enters host cells by utilizing angiotensin-converting enzyme
2 (ACE2), an enzyme which is also a target for treatment of hypertension.
The described co-amorphous solids of VAL may contribute to establishing
a new perspective in the hypertension treatment, in which in addition
to taking anti-hypertensive drugs (VAL), patients will also support
their anti-viral immune response (NIC). Moreover, the anxiety-reducing
potential of NIC is particularly important in COVID-19 pandemic associated
with an increased risk of mental disorders, which can potentially
compromise blood pressure control. It
has been demonstrated that applying FT-IR analysis assisted by DFT,
QTAIM, and NCI calculations in the field of co-amorphous solid dispersions
allows us to estimate what kind of intermolecular interactions occurs
in the pharmaceutical amorphous, solid forms. Analysis of VAL/NIC
systems via the FT-IR technique confirmed that strong
hydrogen bonds between the two components play a significant role
in their stability, while QTAIM and NCI calculations provided a clear
insight into the stability mechanism at the molecular level. The described
approach revealed the undeniable importance of hydrogen bonds between
VAL and NIC in the formation of co-amorphous compositions. These interactions
cause important changes in the arrangement of the VAL tetrazole ring,
which results in the reduced participation of the tetrazolyl N–H
donor and thus reduced co-crystallization. Therefore, the proposed
methodology can be used as a general approach to the design of the
desired co-amorphous solid materials including drugs. Based on the
analyses performed, it can be concluded that co-amorphous solid dispersions
obtained by the solution- and solid-state approaches involved different
kinds of intermolecular interactions, which also resulted in a slightly
different solubility of the dispersions studied. Moreover, it can
be assumed that the solution (slurry) and ball mill techniques provide
more homogenous co-amorphous solid dispersions than the liquid-assisted
grinding. 

