Molecular Pharmaceutics
Article
used more effectively against COVID-19, as well as other
emerging infectious diseases.
reagents were of analytical grade and were commercially
available. They were used without further purification.
Animals. Female BALB/cAJc1 mice (6−7 weeks old, 19
4 g) are considered a suitable model for evaluating the oral
absorption of drugs. Mice were purchased from CLEA Japan,
Inc. and housed in a specific pathogen-free facility under
natural light/dark conditions (temp.: 25 2 °C and relative
humidity: 60 10%) with free access to food and water. The
mice were cared for and handled according to a protocol
approved (A21-279-0) by the Animal Ethics Committee of
Kyushu University, Japan.
To address the abovementioned limitations, an ionic liquid
(IL)-based formulation of FAV is a potential approach to
deliver the drug. ILs have been used extensively in drug
formulations because of their favorable physicochemical and
biopharmaceutical properties compared with crystalline or
other solid forms of drugs.8−11 IL-based active pharmaceutical
ingredient (API) formulations can also address the issue of
polymorphism, which is a problem in modern medicine.9,12
The combination of poorly water-soluble crystalline APIs with
an appropriate IL-forming counterion is a promising technique
to convert conventional pharmaceuticals to an IL form (API-
ILs).13 This technique can reduce the issues of drug
polymorphism and crystallinity, which are often responsible
for the limited aqueous solubility, therapeutic efficiency, and
thermal stability of drugs.14−17 Recently, Samir et al. have
demonstrated the successful application of choline and geranic
acid-based IL formulations to orally deliver sorafenib and
reported improved PK profiles with a 2-, 2.2-, and 1.6-fold
higher drug elimination half-life, peak blood concentration, and
mean absorption time, respectively, compared with the control
formulations.18 In another study, an IL-based formulation
developed for the oral administration of macromolecule insulin
resulted in a 10-fold enhancement of paracellular transport,
with decreases in sustained blood glucose (up to 45%),
compared with insulin injection.19 A sulfasalazine-based IL
formulation also exhibited 4000-fold higher solubility and 2.5-
fold higher bioavailability compared with the parent drug.20
These IL-based oral delivery systems clearly demonstrated that
developing an IL formulation as a new “green” and designable
solvent-based FAV formulation would be a promising method
for improving the therapeutic efficacy. To the best of our
knowledge, there have been no reports that address the existing
constraints with the solid salts of FAV.
Synthesis of FAV-ILs. The API-ILs of FAV were
synthesized using the neutralization method, according to a
previously published procedure.21 The synthetic route for the
AAEs is shown in Scheme S1; an excess amount of thionyl
chloride was added to amino acid in ethanol (mol ratio of
thionyl chloride: amino acid = 1.5:1), followed by neutraliza-
tion by the addition of an ammonium solution (mol ratio of
ammonia to AAE salt = 2:1) in diethyl ether. The synthetic
route for cholinium hydroxide is outlined in Scheme S2; an
excess amount of silver oxide was added to choline chloride in
methanol.22 Finally, an equimolar amount of FAV and an AAE
or a hydroxide salt of an IL-forming cation were stirred
thoroughly in methanol at 40 °C for 2 h. The structures and
1
purities of all the FAV-ILs were determined by H NMR
spectroscopy (JEOL ECZ400S 400 MHz, Tokyo, Japan) and
FT-IR (Frontier FT/IR, Waltham, MA, USA) over the range
PXRD Analysis of the FAV-ILs. To evaluate the structures
of the FAV-ILs, PXRD analysis was carried out using a high-
resolution Rigaku X-ray diffractometer (Tokyo, Japan) with
Ni-filtered monochromatic Cu Kα radiation (λ = 1.5418 Å),
operating at 30 mA and 40 kV. Diffractograms were collected
from a 2θ range of 5−50° and scanned at a speed of 2°/min,
with an angle of 0.02°.
The aim of the present study was to explore the use of the
API-IL technique to solve the deficiencies of crystalline FAV.
FAV was formulated as an anion in a FAV-IL with a series of
IL-forming biocompatible cations, including amino acid ester
(AAE), cholinium (Cho), and quaternary ammonium (TMA)
ions (Figure S1). These cations were selected for their
favorable hydrophilicity and biocompatibility when combined
with the drug. The physicothermal properties of the
synthesized FAV-ILs were investigated by 1H nuclear magnetic
resonance (1H NMR) spectroscopy, Fourier-transform infrared
(FT-IR) spectrometry, powder X-ray diffraction (PXRD),
thermogravimetric analysis (TGA), derivative thermogravim-
etry (DTG), and differential scanning calorimetry (DSC) to
explore how these properties changed with different cations.
Because of the very low aqueous solubility of FAV, sodium
hydroxide solution was added to prepare the control FAV
solution. The solubility, pharmacokinetics, and biodistribution
profiles of the FAV-ILs were determined to evaluate some of
the determinants of in vivo exposure.
TGA of the FAV-ILs. The TGA thermograms of the FAV-
ILs were collected on a Hitachi TG/DTA 7300 (Tokyo,
Japan). Samples in the range 2−5 mg were placed in an
aluminum crucible and analyzed over 30−400 °C at 10 °C/
min under a constant nitrogen stream at a flow rate of 30 mL/
min. To remove the excess volatile matter and residual
impurities from samples, a 30 min isothermal process was also
conducted at 70 °C.
DSC of the FAV-ILs. DSC thermograms were collected on
a Hitachi DSC X7000 (Tokyo, Japan). Samples (3−5 mg)
were placed on aluminum pans and crimped with a standard
aluminum lid. Three cycles of DSC were recorded for each
sample over a temperature range of −80 to 200 °C at a rate of
5 °C/min under an atmosphere of nitrogen at a flow rate of 30
mL/min. An empty aluminum pan with an aluminum lid was
used as a reference. Tg, phase transition, and Tm temperatures
were recorded as the temperature at the midpoint of the
relevant peaks.
Solubility of the FAV-ILs. An excess amount of each FAV-
IL was mixed with 0.5 mL of water, and the mixture was
vigorously stirred at 20 °C for 24 h to evaluate the aqueous
solubility using a “shake-flask” method.23 Then, each solution
was centrifuged at 12,000 ×g for 45 min to facilitate the
separation of the solid and liquid phases. The residues were
removed using a 0.2 μm syringe filter, and the filtrate was
diluted with the mobile phase (acetonitrile: water: acetic acid =
57:43:0.03). The peak area of the obtained solution was
measured at 360 nm after high-performance liquid chromatog-
EXPERIMENTAL SECTION
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Materials. FAV (anhydrous, >98% purity) was purchased
from Manchester Organics Ltd. (Runcorn, UK). L-Proline and
β-alanine (with purity >98.0%) were obtained from Wako
Chemicals Ltd. (Osaka, Japan). Choline chloride and
tetramethylammonium hydroxide (15% in water) were
purchased from Kishida Chemical Co. Ltd. (Osaka, Japan),
and the concentration was determined via titration. All
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Mol. Pharmaceutics 2021, 18, 3108−3115