Preparation and Transformation of Solid Glass Solutions of Clotrimazole to Nanosuspensions with Improved Physicochemical and Antifungal Properties

Abstract

PurposeEvaluating the feasibility of two-step preparation of clotrimazole solid glass solutions for improving its physicochemical properties, intrinsic dissolution, and antifungal activity.Methods
Co-grinding with selected coformers including vitamin C and L-arginine was employed to form co-amorphous dispersions; then, a polymeric carrier was added to form a homogenous glass solution. The solid solutions were converted to nanosuspensions after reconstitution and ultrasonication. The dispersions were characterized for equilibrium solubility, intrinsic dissolution, particle size, and zeta potential. Solid state characterization was carried out using differential scanning calorimetry, X-ray powder diffraction, and Infrared spectroscopy. The antifungal activity was evaluated using candida albicans species.ResultsEquilibrium solubility indicated superb increase in clotrimazole solubility (more than 289 times) from glass solutions compared to pure crystalline drug. The intrinsic dissolution data showed 64% ± 0.34 of drug released within 15 min, and complete dissolution was obtained in 45 min. The ideal formula showed nanosized particles after dispersion in water (225 nm), optimum zeta potential (20.20 µV), and polydispersity index (0.35). Solid state characterization showed shortened peaks and diffraction lines of the glass solutions compared to parent components. The biological activity of the reconstituted nanosuspension showed decreased minimum inhibitory concentration by 50% and increased area of growth inhibition zones of candida albicans by more than 28% compared to drug solution.Conclusions
These findings suggest that solid glass solutions and its derived nanosuspensions of clotrimazole with ascorbic acid and polyvinyl pyrrolidone could be a valuable solution for maximizing drug bioavailability.

Full text

Vol.:(0123456789)
1 3
Journal of Pharmaceutical Innovation
https://doi.org/10.1007/s12247-021-09595-w
ORIGINAL ARTICLE
Preparation andTransformation ofSolid Glass Solutions
ofClotrimazole toNanosuspensions withImproved Physicochemical
andAntifungal Properties
AhmedM.AbdelhaleemAli1 · MusarratHusainWarsi1· MohammedA.S.Abourehab2,3 · AdelAhmedAli4
Accepted: 16 October 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022
Abstract
Purpose Evaluating the feasibility of two-step preparation of clotrimazole solid glass solutions for improving its physico-
chemical properties, intrinsic dissolution, and antifungal activity.
Methods Co-grinding with selected coformers including vitamin C and L-arginine was employed to form co-amorphous
dispersions; then, a polymeric carrier was added to form a homogenous glass solution. The solid solutions were converted
to nanosuspensions after reconstitution and ultrasonication. The dispersions were characterized for equilibrium solubility,
intrinsic dissolution, particle size, and zeta potential. Solid state characterization was carried out using differential scan-
ning calorimetry, X-ray powder diffraction, and Infrared spectroscopy. The antifungal activity was evaluated using candida
albicans species.
Results Equilibrium solubility indicated superb increase in clotrimazole solubility (more than 289 times) from glass solu-
tions compared to pure crystalline drug. The intrinsic dissolution data showed 64% ± 0.34 of drug released within 15min,
and complete dissolution was obtained in 45min. The ideal formula showed nanosized particles after dispersion in water
(225nm), optimum zeta potential (20.20µV), and polydispersity index (0.35). Solid state characterization showed shortened
peaks and diffraction lines of the glass solutions compared to parent components. The biological activity of the reconstituted
nanosuspension showed decreased minimum inhibitory concentration by 50% and increased area of growth inhibition zones
of candida albicans by more than 28% compared to drug solution.
Conclusions These findings suggest that solid glass solutions and its derived nanosuspensions of clotrimazole with ascorbic
acid and polyvinyl pyrrolidone could be a valuable solution for maximizing drug bioavailability.
Keywords Ascorbic acid· Clotrimazole· Intrinsic dissolution· Glass solution· L-arginine· Nanosuspension
* Ahmed M. Abdelhaleem Ali
a.mali@tu.edu.sa; ahmed.mahmoud3@yahoo.com
Musarrat Husain Warsi
mvarsi@tu.edu.sa
Mohammed A. S. Abourehab
maabourehab@uqu.edu.sa
Adel Ahmed Ali
adelahmed.ceutics@yahoo.com
1 Department ofPharmaceutics andIndustrial Pharmacy,
College ofPharmacy, Taif University, P. O. Box11099,
Taif21944, SaudiArabia
2 Department ofPharmaceutics, Faculty ofPharmacy, Umm
Al-Qura University, Makkah21955, SaudiArabia
3 Department ofPharmaceutics andIndustrial Pharmacy,
Faculty ofPharmacy, Minia University, Minia, Egypt
4 Department ofPharmaceutics andIndustrial Pharmacy,
Faculty ofPharmacy, Beni-Suef University, Beni-Suef, Egypt

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Introduction
Numerous drug delivery systems are recently developed for
enhancing physicochemical properties of active pharmaceuti-
cal ingredients (API). The research and development invest-
ments made in multinational pharmaceutical companies on
new drug moieties brought to the market between 2014 and
2018 reached more than US$980 million per drug on average
[1]. This means that a lot of money is spent on innovating
new drugs while very small amounts are spent on developing
existing drug molecules to improve their physicochemical
properties, bioavailability, and reduce the costs of manufac-
ture. Expensive drug delivery systems involve nanoparticle
design using specific lipids and surfactants or complicated
vesicular systems which are often suffering from physical
instability. Therefore, researchers are pushed to develop new
and costless drug delivery approaches for getting the same
benefits without paying too much time or money.
One of the most popular, fast, and cheap techniques used
for drug development are solid dispersions that aim at trans-
forming crystalline drugs into amorphous, co-amorphous, or
semicrystalline counterparts in order to improve their solu-
bility and hence bioavailability [2]. Some of these disper-
sions are based on using hydrotropic molecules such as low
molecular weight carboxylic acids [3], amino acids [4], and
others depend on hydrophilic large molecules such as hydro-
philic polymers [5]. Some recently applied solid dispersion
techniques involve H-bonding donor/acceptor interactions
between the parent drug and a small molecule as a coformer
such as using glucosamine with Paracetamol [6], nicotina-
mide, and quetiapine to form co-amorphous dispersions and
also for possibility of forming cocrystals [7].
Clotrimazole (CLT) is a broadspectrum antifungal drug
with low solubility (0.49mg/L) [8] and high permeability;
hence, it is classified as BCS class II drug [9]. Cases of fun-
gal infections are continuously increasing especially in low
immunity patients. This is usually attributed to excessive use of
antibiotics and glucocorticoids. Therefore, various antimycotic
agents formulated into fast release formulations are highly
needed to overcome different cases of mycosis. There are many
research studies that deal with solving the physicochemical
properties of clotrimazole to strengthen its efficacy and bioa-
vailability including formulation with chitosan-monoglyceride
nanoparticles [10]. Formulation of clotrimazole with essential
oils was undertaken to enhance its antifungal activity [11].
Solid glass solutions are effective technique that is used to
transform crystalline drugs into stable amorphous form with
hydrophilic carriers and avoid phase separation or recrystal-
lization with maximized solubility and dissolution rate [12].
Successful examples include indomethacin with PVP which
was prepared using fluidized bed coating [13]. Celecoxib
and PVP were also prepared into glass solution using holt-
melt extrusion [14].
However, large amounts of carriers lead to high volume
of final solid dosage form and sometimes the hygroscopic
nature of the polymer adds another difficulty to manufacture
[15].
In this paper, preparing a solid glass solution of clotri-
mazole based on co-amorphization with a small molecule
(ascorbic acid or L-arginine) by co-grinding in presence of
small amount of a hydrophilic carrier will be addressed. The
produced modified glass solutions will be reconstituted to a
nanosuspension to explore its liquid dispersion properties.
It is expected that this improved technique will encompass
added benefits of improving drug physicochemical proper-
ties, formulation into liquid form, and hence maximizing its
bioavailability.
Materials andMethods
Materials
Clotrimazole (Lot. No. BCBQ4635) and Hydroxy Propyl
Methyl Cellulose viscosity grade 2.6–5.6 cP at 2% (w/v)
concentration (Lot No. MKBZ3047V) were procured from
Sigma Aldrich CO., St. Louis, USA. L-arginine (Lot. No.
K32444744406) was purchased from VWR International
Ltd., Poole, England. Ascorbic acid was bought from HIME-
DIA Labs. PVT. Ltd. Mumbai, India. Pluronic F127 and
crosslinked polyvinyl pyrrolidone were purchased from
ACROS Organics, New Jersey, USA. All other reagents
were of analytical grade. Acetonitrile HPLC grade (Lot No.
1711910) was purchased from Fischer Scientific, Loughbor-
ough, UK. Orthophosphoric acid was obtained from LOBA
CHEMIE PVT. Ltd. Mumbai, India.
Fig. 1 Chemical structure of clotrimazole and two coformers Ascor-
bic acid and L-arginine

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Preparation ofClotrimazole Solid Dispersions
Solid dispersions of clotrimazole with selected coformers
from amino acids namely L-arginine (LA) and hydrotropic
carboxylic acids namely ascorbic acid (ASC) (Fig.1) were
performed using two-step co-grinding method. According
to drug to coformer molar ratio data in Table1, the weighed
amounts of the drug and coformer were transferred to a
small porcelain mortar and ground together for 15min.
Then, a fixed amount (50mg) of a carrier polymer namely
hydroxypropyl methyl cellulose (HPMC), Pluronic F127, or
crosslinked polyvinyl pyrrolidone was added to the mixture
and co-grinding was continued for another 5min. The car-
rier polymer served for stabilizing the amorphous state of
the glass solution and the nanosuspension after being dis-
persed in water.
Quantification ofDrug
HPLC Method
First a calibration curve was constructed for clotrimazole
based on a previous method with modification [16]. The
mobile phase consisted of acetonitrile:water (65:35) with
pH 3.0 adjusted with orthophosphoric acid. Briefly, ten mil-
ligrams of drug were dissolved in 100mL of mobile phase;
then, serial dilutions were made resulting in a series of con-
centration range 10–50µg/mL. Finally, 20 µL samples from
each concentration were injected on the system. The HPLC
system was Shimadzu HPLC, C18 Column (Shimadzu,
Japan) operated at flow rate 0.5mL/min, and oven tempera-
ture was kept at 28°C.
Differential Scanning Calorimetry
The differential scanning calorimeter (Model STA 449 F3
Jupiter, Nietzsche, Germany) was operated for solid state
characterization of prepared solid dispersion under an
atmosphere of nitrogen. Prepared solid dispersions (5mg)
were placed in small aluminium pans, covered with alu-
minium lids, and heated at a rate of 10°C/min. The range
of heating temperature was set between 20 and 300°C [17].
Fourier Transform Infrared
Samples (5mg) of clotrimazole and coformers; L-arginine,
ascorbic acid, and their physical mixtures and the prepared
solid dispersions were separately mixed with 400mg dry
potassium bromide. Then, the powder mixtures were com-
pressed into small discs using a hydrostatic press [18]. A
Fourier transform infrared instrument (IR Prestige-21, Shi-
madzu, Japan) was used to capture the infrared spectrum at
a scanning range of 400–4000 cm−1.
X‑ray Powder Diffraction
Samples of pure drug and coformers as well as solid disper-
sions and physical mixtures were characterized for crystal-
linity using X-ray diffraction analysis (Shimadzu XRD-6000
X-ray powder diffractometer, Shimadzu, Japan). The XRD
system was equipped with a standard Cu sealed X-ray tube
operated at 40kV voltage and 40mA current. Data acquisi-
tion was performed at 2-Theta angle range of 5–60° with
0.04 steps and scanning speed of 0.4°/s [19].
Table 1 Composition of clotrimazole solid dispersion formulations
Mr* molar ratio, cPVP crosslinked polyvinyl pyrrolidone
Formulation Drug (50mg) Coformer Carrier polymer
(50mg)
Amorphization method Drug to cofor-
mer Mr* % w/w drug
1 Clotrimazole Vitamin C HPMC Co-grinding 1:01 39.80
2 Clotrimazole Vitamin C PF127 Co-grinding 1:01 39.80
3 Clotrimazole Vitamin C cPVP Co-grinding 1:01 39.80
4 Clotrimazole L-Arginine HPMC Co-grinding 1:01 39.90
5 Clotrimazole L-Arginine PF127 Co-grinding 1:01 39.90
6 Clotrimazole L-Arginine cPVP Co-grinding 1:01 39.90
7 Clotrimazole Vitamin C HPMC Co-grinding 1:02 33.10
8 Clotrimazole Vitamin C PF127 Co-grinding 1:02 33.10
9Clotrimazole Vitamin C cPVP Co-grinding 1:02 33.10
10 Clotrimazole L-Arginine HPMC Co-grinding 1:02 33.20
11 Clotrimazole L-Arginine PF127 Co-grinding 1:02 33.20
12 Clotrimazole L-Arginine cPVP Co-grinding 1:02 33.20

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Determination ofEquilibrium Solubility
oftheDispersions
Samples from the prepared solid dispersions (5mg) and the
pure drug were placed in Eppendorf tubes with 1mL of pure
distilled water. Samples were continuously shaken using a
Stuart mini-orbital shaker (UK) for 3h. Aliquots of 200 µL
were withdrawn and then diluted to 5mL with water fol-
lowed by filtration using a 0.45 µm disc filter. Before injec-
tion, 750 µL filtered samples were taken and diluted with
equal amount of mobile phase. The concentrations (µg/mL)
of the injected samples were calculated from the previously
constructed calibration data, and statistical comparisons
were made between results of test formulation and pure
clotrimazole.
Determination oftheIntrinsic Dissolution
Tablets (10mg) were prepared inside metal discs using mini
hand press (Shimadzu Japan). The solid dispersion and the
pure drug were individually placed on the top of the discs
and pressed into tablets. One side of the disc was covered
with molten Beeswax, and other side was exposed to the dis-
solution media [20]. Five discs (containing five compressed
tablets) were weighed before and after compression to obtain
the net weight of the compressed powder. The medium con-
sisted of 250mL of 0.1 normal HCl (pH 1.2) using paddle
rotating at 50rpm and maintained at 37 ± 0.5 using coil-
heated water path of Logan Dissolution Apparatus (New
Jersey, USA). Samples were withdrawn at 15, 30, 60, and
90min, filtered through a 0.45 µm filter then diluted with
mobile phase before HPLC analysis [19]. Differences in dis-
solution results between test and standard tablets were evalu-
ated statistically at a level of significance P = 0.05.
Reconstitution oftheGlassy Dispersions
The prepared solid glass solutions by co-grinding of Clotrima-
zole with the small molecules viz ascorbic acid or L-arginine
in the presence of hydrophilic polymers were tested for pos-
sibility of forming supersaturated solutions or nanosuspen-
sions after reduction of particle sizes during comminution.
Samples of 5mg from each dispersion were reconstituted with
10mL of pure water and subjected to high shear ultrasonica-
tion (Ultrasonication bath-Branson, 3510, USA) for 2min. A
total of 100 µL were withdrawn and diluted to 1mL with pure
water before measuring PS and ZP using Malvern Zetasizer
(Zeta sizer nano series-Malvern, UK).
Determination oftheAntifungal Activity
Antimicrobial activity test using agar diffusion method (Cup
Technique) and minimum inhibitory concentration (MIC)
determination was carried out on formulation showing high-
est equilibrium solubility and intrinsic dissolution rate (F9).
Test samples were measured and compared to the pure drug
for the antifungal activity on candida albicans species.
Fig. 2 DSC of single components including L-arginine (A), HPMC (B), ascorbic acid (C), PVP (D), Pluronic F127 (E), and Clotrimazole (F)

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Fig. 3 DSC of clotrimazole
(1:1) physical mixtures with
vitamin C (A), L-arginine (B),
HPMC (C), PF127 (D), and
PVP (E)

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The cup plate method is one of the official methods,
where the test samples diffuse from the cup through an agar
layer in a Petri dish to such an extent that the growth of
added microorganisms (Candida albicans ATCC 60,913) is
restricted entirely to a circular area or zone around the cav-
ity containing the solution of an antibiotic substance [21].
The antimicrobial activity was expressed as zone diameter
in millimetres, which is measured by a scale [22].
All samples (standard and test) were dissolved to give
final concentrations of 1000, 200, 100, 50, 25, 10, 5, and
1µg/mL.
An overnight culture of the tested microorganism was
mixed with Brain Heart agar media to give a final concentra-
tion of 1% microorganism (about 0.5 McFarland) and poured
into sterile Petri dishes in a fixed amount of 20mL using
aseptic conditions. The sterile cork borer was used to pre-
pare cups. Test samples and standard drugs with volumes of
80 μL were introduced into cups with the help of a micropi-
pette. All the plates were kept at room temperature for effec-
tive diffusion of the test drug and standard and incubated at
30 ± 1°C for 24h. The presence of inhibition zones around
the cup indicated antibacterial activity. The diameter of the
zone of inhibition was measured and recorded [23].
A dose response curve was prepared based on plotting
log concentrations versus diameters to explore the trend of
activity and differentiate between test and standard formu-
lations at different concentrations. The linear regression
equation was generated at which MICs were calculated,
which is the lowest concentration causing complete inhibi-
tion of growth [24].
Fig. 4 DSC of co-ground
glass solutions of clotrimazole
with ascorbic acid (A) and
L-arginine (B) at 1:1 and 1:2
molar ratios (F1-3, F7-9) and
(F4-6, F10-12), in the pres-
ence of HPMC, Pluronic F127,
and polyvinyl pyrrolidone as
carrier polymers as described in
Table1

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Results andDiscussion
Solid State Characterization
DSC
The results of DSC showed a characteristic melting peak
for clotrimazole observed at 152°C as indicated by other
researchers in the literature [25]; ascorbic acid also dem-
onstrated a sharp melting peak at 178°C [18]. L-arginine
showed three characteristic peaks at 100, 224, and 246°C
indicating loss of water, then initial and complete melt-
ing of the two D/L enantiomeric mixtures, respectively
[26–28]. The melting peaks for the carrier excipients
HPMC, Pluronic F127, and crosslinked PVP were broad
and non-sharp indicating amorphous nature of the excipi-
ents (Fig.2).
The DSC thermograms for the physical mixtures
of clotrimazole with coformers and carrier polymers
(Fig.3) showed summation of single peaks for each com-
ponent. The thermogram of clotrimazole with Vitamin
C (Fig.3A) showed a characteristic adjacent melting
(138°C) and recrystallization (152°C) peaks possibly
indicating molecular interactions between the two com-
pounds leading to a new phase having new reduced melt-
ing temperature.
Fig. 5 IR spectra of clotrima-
zole (A) and other coformers:
ascorbic acid (B), L-arginine
(C), HPMC (D), Pluronic F127
(E), and polyvinyl pyrrolidone
(F)
Fig. 6 IR spectra of clotrima-
zole (A) and its 1:1 physical
mixture with ascorbic acid (B),
L-arginine (C), HPMC (D), Plu-
ronic F127 (E), and polyvinyl
pyrrolidone (F)

Journal of Pharmaceutical Innovation
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The thermograms of co-ground glass solution samples
of clotrimazole with vitamin C at 1:1 molar ratio
(Fig.4A, F1, F2, F3) showed complete disappearance
of the melting endotherm of the drug especially with
HPMC and PVP (F1 and F3) with shortened peak in
case of PF127 (F2) appeared at 130°C. In case of
1:2 molar ratio (F7, F8, F9), a similar endothermic
behaviour was obtained with HPMC and PF127, whilst
a new exothermic peak was obtained at 143°C with
PVP (F9) possibly indicating a molecular interaction
and formation of a new phase. The glass solutions pre-
pared with L-arginine (F4B, F5, F6) at 1:1 molar ratio
and those at 1:2 ratio were similar except with PF127
(F5 and F11), where complete disappearance of clotri-
mazole melting peak occurred with the first and only
shortened and broadened in the latter indicating that
the optimum ratio for possible H-bonding interactions
with L-arginine could happen at low molar ratio.
Infrared Spectroscopy
The results of IR investigation for clotrimazole coformers
and carrier polymers as well as glass solution formulations
are shown in Figs.5–8. The characteristic absorption peaks
of clotrimazole are found as the aromatic C-H stretching at
3063 cm−1, C = C stretching at 1587 cm−1, C = N stretching at
Fig. 7 IR spectra of clotrima-
zole (A) and its glass solution
formulations with ascorbic acid
at 1:1 ratio and HPMC (B),
PF127 (C), PVP (D), and at 1:2
ratio in the presence of HPMC
(E), Pluronic F127 (F), and
polyvinyl pyrrolidone (G)
Fig. 8 IR spectra of clotrima-
zole (A) and its glass solution
formulations with L-arginine at
1:1 ratio and HPMC (B), PF127
(C), PVP (D), and at 1:2 ratio in
the presence of HPMC (E), Plu-
ronic F127 (F), and polyvinyl
pyrrolidone (G)

Journal of Pharmaceutical Innovation
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1566 cm−1, the chlorobenzene ring stretching at 1210 cm−1,
and the C-N stretching at 1081 and 1040 cm−1 with C-H
aromatic bending where similar to those found in the lit-
erature [11] [25]. Ascorbic acid showed the common alco-
holic OH stretching at 3479 cm−1 and aliphatic CH stretch-
ing at 3333 cm−1, a characteristic C = O stretching peak at
1762 cm−1 in addition to the C = C double bond stretching
(1674 cm−1), and the peak of -OH stretching observed at
1318 cm−1 which were also similar to previously reported
data [29]. The IR absorption spectra for L-arginine (Fig.5C)
showed characteristic broad -NH stretching and -OH stretch-
ing bands overlapping at 3294 and 3090 cm−1 and charac-
teristic C = O stretching at 1636 and 1638 cm−1 as reported
earlier in the literature [30]. The IR spectra of the 1:1 physi-
cal mixtures (Fig.6) were summation of spectra of individual
components which appeared as shorter peaks due to dilution.
The IR spectra of the solid glass solutions showed overlap
between the C = C aromatic stretching bands of clotrimazole
bands at 1490 and 1433 cm−1 with vitamin C carbonyl group
stretching band at 1474 cm−1 (which was also broadened)
with shortening of all other peaks which possibly indicate
molecular interactions (Fig.7). While the glass solutions
with L-arginine (Fig.8) showed IR spectra of only short-
ened peaks at both molar ratios, no shifting or appearance
of new peaks was observed indicating absence of molecular
interactions.
Results ofXRD
The results of XRD analysis (Fig.9) showed a characteristic pat-
tern of diffraction lines for Clotrimazole, Vitamin C, L-Arginine,
Pluronic F127, HPMC, and PVP. Clotrimazole demonstrated
prominent diffraction lines at 2 Theta of 10.17, 12.50, 14.20. 16.
86, 18.60, 19.58. 20.70, 23.16. 25.19, and 28.21° as reported in
the literature for crystalline clotrimazole [31].
Ascorbic acid demonstrated numerous sharp diffraction
lines at 2 Theta 12.21, 19.60, 19,75, 24.39, 25.23, 26.61,
29.85, and 33.31° as previously reported [18]. L-arginine
showed four prominent diffraction lines at 2 Theta of 11,
19.29, 23, and 27.3°, while Pluronic F127, Crosslinked PVP,
and HPMC were mostly amorphous polymers or semicrys-
talline powders showing only few short diffraction lines [32].
The diffraction lines for physical mixtures (Fig.10)
showed summation of parent component diffraction lines
and represented a highly crystalline mixture in case of
clotrimazole with ascorbic acid (PM1) and L-arginine
(PM2), whilst physical mixtures with the carrier polymers
HPMC, PVP, and Pluronic F127 (PM3-5) showed very
short and non-sharp diffraction lines indicating dilution
of clotrimazole inside the amorphous polymer.
Most of the diffraction lines shown for the solid glass
solutions (Fig.11) demonstrated very short and non-sharp
lines indicating highly reduced crystallinity of clotrimazole
and coformers in the prepared dispersions (in case of F1,
F2, F3, F8, and F9 with ascorbic acid). This could be due
to partial amorphization of the drug through H-bonding
with the coformers in addition to dilution and stabilization
by the carrier polymers. Some dispersions retained some
crystallinity and showed a characteristic sharp single dif-
fraction line at 2 Theta 22.30° possibly due to remaining
crystals of L-arginine (in case of F5, F6, F10, F11) and
ascorbic acid (F7).
Fig. 9 XRD diffraction lines of clotrimazole and single coformers (L-arginine and Vitamin C) and polymeric carriers (Pluronic F127, HPMC,
and PVP)

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Fig. 10 XRD diffraction lines of
clotrimazole 1:1 physical mix-
tures with ascorbic acid (PM1),
L-arginine (PM2), HPMC
(PM3), Pluronic F127 (PM4),
and PVP (PM5)

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Results ofEquilibrium Solubility
From the equilibrium solubility data shown in Table2,
it was noted that the highest solubility (598.8µg/mL)
was obtained from Formula F9 composed of clotrima-
zole and ascorbic acid at 1:2 molar ratio with polyvinyl
pyrrolidone as the carrier polymer; the second formula is
F8 (535.65µg/mL) containing the same composition of
clotrimazole and ascorbic acid but with the added carrier
polymer Pluronic F127. The data indicates that glass solu-
tions of clotrimazole with ascorbic acid have significantly
higher percentage solubility (P = 0.001) compared to those
with L-arginine at both molar concentrations 1:1 and 1:2
and in the presence of each type of carrier polymer (as
demonstrated in Fig.12). The data also indicated more
than 280 times increase in solubility of clotrimazole from
the glass solution F9 compared to the pure drug. These
findings and specifically the highest solubility imparted
by inclusion of PVP in the glass solution were similar to
other studies reported in the literature for solid dispersions
of clotrimazole with the same polymer [33].
Results ofIntrinsic Dissolution
Disc tablets (n = 5) prepared from solid glass solutions (F9)
showed high percentage of drug dissolved (64%) within the
first 15min and reached 100% within 45min compared to
pure clotrimazole which gave only 43% and 84.5% within
the same time periods, respectively (Table3). Similar results
were obtained from studies addressed improvement of clotri-
mazole dissolution using solid dispersions with Pluronic
F127 prepared by fusion and co-grinding [34]. Statistical
analyses of the results of dissolution showed significant dif-
ferences between F9 and clotrimazole disc tablets at 15 and
45min with a P-value = 0.001 and 0.008, respectively.
Transforming theGlass Solution intoNanosuspension
Samples of the prepared glass solutions were assessed for
nanoparticle size (PS) and zeta potential (ZP) after dis-
persion in pure water. This experiment was undertaken
to explore correlation between glass solution solid state,
physicochemical properties, and final dispersion particulate
properties. It is expected that the dispersions which were
subjected co-grinding have developed semicrystalline (fine
crystals), amorphous or co-amorphous dispersions of clotri-
mazole. Although ordinary amorphous combinations require
large amounts of carrier polymers [35], the new strategy
Fig. 11 XRD diffraction lines of clotrimazole-coformer co-ground glass solutions (F1-F12)
Table 2 Equilibrium solubility of prepared solid dispersions
Formula Theoretical
amount (µg/
ml)
Amount dis-
solved (µg/
mL)
Percentage
dissolved
No. of times
increase in
solubility
F1 1990.0 129.34 6.50 62.5
F2 2149.2 285.16 13.27 137.8
F3 1990.0 440.37 22.13 212.70
F4 1995.0 1.77 0.09 0.90
F5 1995.0 63.01 3.16 30.40
F6 2074.8 2.36 0.11 1.10
F7 1820.5 343.51 18.87 165.90
F8 1655.0 535.65 32.37 258.80
F9 1820.5 598.79 32.89 289.30
F10 2091.6 2.60 0.12 1.30
F11 1826.0 52.85 2.89 25.50
F12 1759.6 2.30 0.13 1.10
Drug 6100.0 2.07 0.03 -

Journal of Pharmaceutical Innovation
1 3
of adding a coformer with small amount of carrier to form
glassy dispersions might be effective in reducing the dose
size. Also, the produced dispersions can be converted eas-
ily to a supersaturated and stabilized nanosuspension form
brought about by the added small amounts of hydrophilic
carrier polymers as previously reported in the literature for
similar amorphous dispersions [36]. It was noticed from the
data in Table4 that the lowest PS (225nm) was demon-
strated by F9 with lowest poly dispersity index (0.35). The
zeta potential was moderately reduced compared to that of
pure clotrimazole particles. The above findings support the
idea that glass solutions prepared by co-grinding and show-
ing co-amorphous drug coformer combination could be
transformed to stable nanosized particles or supersaturated
nanosuspensions especially in presence of a hydrophilic car-
rier during co-grinding.
Antifungal Activity
The zones of inhibition against concentration graph show a
prominent difference between the test formulation and the
standard drug, where high efficacy of the solid dispersion
formulation was detected specially at high concentration
Fig. 12 Percentage equilibrium
solubility of clotrimazole glass
solutions (F1–F12) compared to
pure clotrimazole
Table 3 Intrinsic dissolution of clotrimazole from solid glass solution
(F9) compared to pure clotrimazole
Time (min) Percentage dissolved ± SD
Clotrimazole Solid glass
solution
(F9)
15.0 43.0 ± 0.23 64.0 ± 0.34
30.0 68.0 ± 0.42 96.0 ± 0.54
45.0 84.5 ± 0.31 100.0 ± 0.18
Table 4 Glass solution-transformed nanosuspension properties
Sample name Z-Ave (nm) PDI Zeta
potential
(µV)
F7 356 1.00 −0.77
F8 348 0.43 0.60
F9 225 0.35 20.20
Clotrimazole 3462 1.00 26.00
Table 5 The zones of inhibition
for Candida albicans measured
by a scale in millimetres
M.O Concentration
(µg/mL) Standard (CLT) Test (F9) % increase in the
inhibition zone
Candida albicans ATCC 60,913 1 22.5 23.0 2.22
5 25.0 25.5 2.00
10 25.5 27.0 5.88
25 26.0 28.0 7.69
50 26.5 28.5 7.54
100 27.0 29.0 7.40
200 27.5 30.0 5.45
1000 28.0 36.0 28.57

Journal of Pharmaceutical Innovation
1 3
levels (Table5). The minimum inhibitory concentration
(MIC) was determined to be 0.34 and 0.15µg/mL for stand-
ard (clotrimazole) and test formula (F9), respectively. The
zones of inhibition shown in Fig.13 and Table5 were found
to be increasing at a higher rate for the new glass solution
formula (F9) for concentrations from 1 to 25µg/mL. The
maximum increase in the inhibition zone reached more than
28% at 1000µg/mL compared to pure drug having the same
concentration. The differences in the zones of inhibition
were statistically significant only at a concentration of 1mg/
mL (P-value = 0.01).
Conclusion
The prepared glass solutions by two-step co-grinding
of clotrimazole and the coformer ascorbic acid at 1:1
molar ratio and carrier polymers either Pluronic F127 or
crosslinked polyvinyl pyrrolidone showed highly improved
physicochemical properties. The solid solutions were suc-
cessfully converted to nanosuspensions with improved solu-
bility and intrinsic dissolution possibly due to H-binding
interactions of the drug and coformer leading to reduced
crystallinity and formation of the co-amorphous state. The
solid and liquid states of the dispersions were stabilized by
the added carrier polymer. These findings confirm that glass
solutions of poorly soluble drugs could be transformed to
stable nanodispersions with improved physicochemical and
biological properties.
Acknowledgements The authors of this research would like to
acknowledge the financial support offered by Taif University Research-
ers Supporting Project number (TURSP-2020/50), Taif University,
Taif, Saudi Arabia. The authors would also like to thank the Deanship
of Scientific Research at Umm Al-Qura University for supporting this
work by Grant: 19-MED-1-01-0024 to Mohammed A.S. Abourehab.
Funding The authors of this research received financial support offered
by Taif University Researchers Supporting Project number (TURSP-
2020/50), Taif University, Taif, Saudi Arabia.
Declarations
Conflict of interest The authors declare no competing interests.
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