Cubosomes as Oral Drug Delivery Systems: A Promising Approach for Enhancing the Release of Clopidogrel Bisulphate in the Intestine

Abstract

Clopidogrel bisulphate (CB) is a first line antiplatelet drug for treatment of myocardial infarction and stoke. Yet, its efficacy is limited by its poor solubility in intestinal pH, its main site of absorption. The main aim of this study is to enhance the intestinal release of CB by loading in cubosome nanoparticles. Glyceryl monooleate (GMO) based CB loaded cubosomes were prepared using a 3³ factorial design to study the effect of polyvinyl alcohol (PVA), poloxamer 407 (PL407) concentrations and ratio of CB to the disperse phase on the average particle size, entrapment efficiency (%EE), in vitro release at 15 minutes (%Q15), and their morphology using TEM. The release of the optimized formula was compared in buffer transition media (pH1.2 for 2 hours then pH 6.8 for 6 hours) to free drug to study the effect of the changing pH in the GIT on CB release. The antihaemostatic properties of the optimized formula were compared to the commercial product Plavix® using bleeding time (BT) model in rabbits. The prepared cubosomes were in the nano range (115±6.47 to 248±4.63 nm) with high %EE (91.22±4.09% to 98.98±3.21%). The optimized formula showed significantly higher (p<0.05) CB release in intestinal pH and preserved the high% released (95.66±1.87%) in buffer transition release study compared to free drug (66.82±4.12%) as well as significantly (p<0.05) higher antihaemostatic properties with longer BT (628.47±6.12 seconds) compared to Plavix® (412.43±7.97 seconds). Thus, cubosomes proved to be a successful platform to enhance the intestinal release of CB and improve its absorption.

Full text

© 2018 The Pharmaceutical Society of Japan
Vol. 66, No. 12 1165Chem. Pharm. Bull. 66, 1165–1173 (2018)
Regular Article
Cubosomes as Oral Drug Delivery Systems: A Promising Approach for
Enhancing the Release of Clopidogrel Bisulphate in the Intestine
Hanan M. El-Laithy,a,b Alia Badawi,a Nevine Shawky Abdelmalak,a and Nihal El-Sayyad*,b
a Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University; Cairo 11562,
Egypt: and b Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, October University for
Modern Sciences and Arts (MSA); 6th of October 12582, Egypt.
Received August 9, 2018; accepted September 6, 2018; advance publication released online September 19, 2018
Clopidogrel bisulphate (CB) is a first line antiplatelet drug for treatment of myocardial infarction and
stroke. Yet, its efficacy is limited by its poor solubility in intestinal pH, its main site of absorption. The main
aim of this study is to enhance the intestinal release of CB by loading in cubosome nanoparticles. Glyceryl
monooleate (GMO) based CB loaded cubosomes were prepared using a 33 factorial design to study the effect
of polyvinyl alcohol (PVA), poloxamer 407 (PL407) concentrations and ratio of CB to the disperse phase on
the average particle size, entrapment efficiency (%EE), in vitro release at 15 min (%Q15), and their morphol-
ogy using transmission electron microscopy (TEM). The release of the optimized formula was compared
in buffer transition media (pH 1.2 for 2 h then pH 6.8 for 6 h) to free drug to study the effect of the chang-
ing pH in the gastrointestinal tract (GIT) on CB release. The antihaemostat ic properties of the optimized
formula were compared to the commercial product Plavix® using bleeding time (BT) model in rabbits.
The prepared cubosomes were in the nano range (1156.47 to 2484.63 nm) with high %EE (91.224.09%
to 98.983.21%). The optimized formula showed significantly higher ( p<0.05) CB release in intestinal
pH and preserved the high% released (95.661.87%) in buffer transition release study compared to free
drug (66 .824.12%) as well as significantly (p<0.05) higher antihaemostatic properties with longer BT
(628.476.12 s) compared to Plavix® (412.437.97 s). Thus, cubosomes proved to be a successful platform to
enhance the intestinal release of CB and improve its absorption.
Key words clopidogrel bisulphate; cubosome; factorial design; nanoparticle; buffer transition release study;
bleedi ng time
Clopidogrel bisulphate (CB) is a thienopyridene class anti-
platelet oral drug for the treatment of patients with a high risk
of myocardial infarction and stroke.1) CB is a prodrug that is
absorbed in the intestine and activated in the liver.2) It is ab-
sorbed rapidly following oral administration, with mean half-
life of active metabolite in a range of 7–10 h.3) Clopidogrel is
an inactive prodrug which undergoes CYP3A4-dependent me-
tabolism before exhibiting its antiaggregatory effect.4) CB acts
by selectively and irreversibly inhibiting ADP-induced platelet
activation and aggregation via blocking the binding of ADP to
the purinergic P2Y12 receptor located on the platelet surface.5)
CB is categorized as biopharmaceutics classification sys-
tem (BCS) class II drug, this is primarily due to its poor
solubility in intestinal pH6) the main site of its absorption7)
resulting in very low (<50%) oral bioavailability.8) Following
oral administration, the high solubility of CB in gastric pH,
causes its protonation and ionization and thus hampers its
absorption from the upper gastrointestinal tract (GIT).6) Once
in the intestine, its poor solubility in intestinal pH causes it
to precipitate and consequently diminishes its absor ption and
bioavailability7) Accordingly, if the intestinal solubility and
the release properties of CB is improved, its absorption and
consequently its in vivo bioavailability will be enhanced.9)
Lipid based nanoparticles are considered to be one of the
most successful strategies to enhance the dissolution and per-
meability of drugs. The dr ug is dissolved, entrapped, encapsu-
lated or attached to a nanoparticle matrix thereby remarkably
influencing release profiles of drugs.10) Amongst these drug
delivery systems are cubosome nanoparticles. Cubosomes
are unique and intriguing self-assembled nanoparticles with
enormous potential in diverse areas as medicine, materials
science, and consumer products. Cubosomes are formed by
amphiphilic or surfactant like molecules which self-assemble
into the complex three-dimensional cubic phase str ucture
through the self-organization into bilayers around bicontinu-
ous non-intersecting water channels11) and these structures can
be harnessed to encapsulate hydrophilic, hydrophobic and
amphiphilic molecules.12) They also possess excellent loading
proper ties,13) facilitate absorption14,15) and offer protection for
drugs against degradation16,17 ) thus making them an excel-
lent choice for formulation of poorly soluble drugs orally.18,19)
Cubosomes can maintain the drug in a solubilized state in
the gastrointestinal tract by entrapping drugs into the mixed
micelles produced by the digestion of cubosomes, thereby
enhance drug release and absorption leading to improved oral
bioavailability.13,20)
Therefore, based on the above considerations, the main
aim of this work is to enhance the solubility and the in vitro
release of CB through the formulation of oral cubosome
nanoparticles. A 33 full factorial design was applied to study
the effects of different formulation factors on particle size,
entrapment efficiency (%EE) and percentage of CB release at
15 min in intestinal pH (%Q15) of the resultant formulas. The
pharmacological effect of the optimized formula was assessed
by comparing its antihaemostatic properties through bleed-
ing time (BT) measurements in rabbits to the market product
Plavix® as an indicator of improved CB levels in blood stream
and enhanced drug efficacy.
* To whom correspondence should be addressed. e-mail: nihal_elmahdy@hotmail.com; nmahdy@msa.eun.eg

1166 Vol. 66, No. 12 (2018)
Chem. Pharm. Bull.
Experimental
Materials Clopidogrel bisulfate (CB) was obtained as a
gift from Eva pharma, Egypt. Glyceryl monooleate (GMO),
poloxamer 407 (PL407), polyvinyl alcohol (PVA) were pur-
chased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Spectra/
Pore dialysis membrane (12000–14000 molecular weight cut-
off) was purchased from Spectrum Laboratories Inc. (U.S.A.).
The commercial tablet Plavix® (Sanofi, France) was purchased
to be used in comparative studies. All other chemicals used
were of analytical grade and were obtained from standard
commercial suppliers.
Determination of CB Solubility in Different pH Media
Excess amount of CB was mixed with 100 mL of each of HCl
buffer (pH 1.2), HCl buffer (pH 2.0), phosphate buffer (pH
5.5), phosphate buffer (pH 6.8), phosphate buffer (pH 7.4) and
phosphate buffer (pH 6.8) with 1% Sodium Lauryl Sulphate
(SLS). The solutions were sonicated for 48 h at 37±2°C. The
resulting solutions were left for 24 h to allow excess amounts
to precipitate. The non-dissolved drug was filtered and the
supernatant was analyzed by HPLC method21) using an Agi-
lent 1100 HPLC system (Agilent, U.S.A.) with a UV detector
(Agilent VWD G1314A, U.S.A.). Chromatographic separation
was carried out on Nova-Pak C18 column (5 µm, 4.6×150 mm,
Waters, U.S.A.) maintained at 35°C. The mobile phase con-
sisted of 60% acetonitrile and 40% of 0.05
M sodium hydro-
gen phosphate adjusted to pH 4 using phosphoric acid. The
mobile phase was filtered through 0.2 µm membrane filter and
delivered at a flow rate of 1 mL/min. Samples of 10 µL were
injected and effluents were monitored at 220 nm.
Preparation of CB Loaded Cubosome Dispersions CB
loaded cubosome dispersions were prepared according to the
method described by Morsi et al.22) First GMO and PL407
were melted on a hot plate at temperature 70±2°C. Clopido-
grel was dispersed in the molten mixture. PVA was dissolved
in 2 mL distilled water at 80°C then was added dropwise to
the molten mixture and thoroughly mixed at 1500 rpm with
magnetic stirrer (Thermo Scientific, U.S.A.) till cubic gel was
formed. To form the cubosome dispersions, distilled water
was added dropwise to the cubic gel at 70±2°C under me-
chanical stirring so that the disperse phase (molten mixture)
would constitute 10% from the final dispersion. Dispersions
were maintained under stirring and were cooled to room tem-
perature and then stored in glass bottles at room temperature
for further investigations.
Statistical Design of the Study A 33 full factorial ex-
perimental design was applied to investigate the influence of
the formulation variables such as PL407 concentration (2.5, 5,
10%), PVA concentration (0, 2.5, 5%) as well as the drug to
the disperse phase ratio (1 : 10, 2 : 10, 3 : 10) on the character-
istics of cubosome dispersions (dependent variables) such as
particle size, entrapment efficiency (%EE) and in vitro drug
dissolved at 15 min (%Q15) using Minitab 17® sof tware (ver-
sion 17, Minitab Inc., U.S.A.). The composition of different
formulations (CL1–CL27) is listed in Table 1.
Data collected for each response of the dependent variables
from the 27 runs was analyzed using first order multiple linear
regression model by fitting into the following formula.23)
o ii
YββX=+
(1)
Where Y is the measured response associated with each factor
level combinations, βo is the model intercept, βi is the linear
coefficient and Xi is the level of the independent variable.
The first-order polynomial in Eq. 1 was fitted to raw re-
sponses. ANOVA was used to evaluate the statistical signifi-
cance of the model. Level of significance was set at (α=0.05).
The influence of response variable transformation was investi-
gated by fitting Eq. 1 to log, reciprocal, square root and square
of transformed response variables and comparing two statisti-
cal parameters: the adjusted multiple correlation coefficient
(adjusted R2) and the p values for the overall F-ratio test of
fit adequacy.24) Main effects plots and interaction plots were
obtained and visually inspected.
Additionally, desirability values were calculated for the
optimization and selection of the optimized formula with
the highest desirability value. According to Derringer and
Suich,25) desirability index (D) is a score between 0 and 1
reflecting the level of satisfaction with a given combination of
independent variables. D of 0 corresponds to a completely un-
desirable formulation, and D of 1 corresponds to a completely
desirable one.
If maximization of a response Y is wanted, desirability is
calculated for each Y value according to the following equa-
tion:
min
min max
max min
for
Dy
0 otherwise
YY
Y YY
YY
−

≤≤
−



=
(2)
If minimization of Y is wanted:
Table 1. Composition of Different Prepared CB Loaded Cubosome Dis-
persions
Formula
Disperse phase Ratio of drug to
disperse phase
GMO (%) PL407 (%) PVA (%)
CL1 97.5 2.5 0 1 : 10
CL2 95 2.5 2.5 1 : 10
CL3 92.5 2.5 5 1 : 10
CL4 95 5 0 1 : 10
CL5 92.5 5 2.5 1 : 10
CL6 90 5 5 1 : 10
CL7 90 10 0 1 : 10
CL8 87.5 10 2.5 1 : 10
CL9 85 10 5 1 : 10
CL10 97.5 2.5 0 2 : 10
CL11 95 2.5 2.5 2 : 10
CL12 92.5 2.5 5 2 : 10
CL13 95 5 0 2 : 10
CL14 92.5 5 2.5 2 : 10
CL15 90 5 5 2 : 10
CL16 90 10 0 2 : 10
CL17 87.5 10 2.5 2 : 10
CL18 85 10 5 2 : 10
CL19 97.5 2.5 0 3 : 10
CL20 95 2.5 2.5 3 : 10
CL21 92.5 2.5 5 3 : 10
CL22 95 5 0 3 : 10
CL23 92.5 5 2.5 3 : 10
CL24 90 5 5 3 : 10
CL25 90 10 0 3 : 10
CL26 87.5 10 2.5 3 : 10
CL27 85 10 5 3 : 10

Vol. 66, No. 12 (2018) 1167
Chem. Pharm. Bull.
max
min max
max min
for
Dy
0 otherwise
YY
Y YY
YY
−

≤≤
−



=
(3)
If a target response, Ytarg, is wanted:
targ
targ max
max min
targ
min targ
max min
1 for
Dy
1 for
0 otherwise
YY
Y YY
YY
YY Y YY
YY
−

− ≤≤
−

−
− ≤≤
−



=
(4)
where Ymin and Ymax define ranges of acceptable response
values. When multiple responses are present, individual desir-
ability index must be calculated for each response, then a joint
desirability (Djoint) is defined as the weighted geometric mean
of individual desirability indices normalized by the number of
responses n:
i
i
1/
joint
1
DD
n
n
W
Y
i




∏
=
=
(5)
where Wi are relative importance weights of different respons-
es (the weights must add up to 1).
In this st udy, the criteria set for optimal formulation selec-
tion was achieving the least particle size associated with the
maximum %EE and the highest %Q15. The three criteria were
considered equally important; therefore, the weights were set
to 1/3.
Transmission Electron Microscopy (TEM) One drop
of the cubosome dispersion was deposited on carbon-coated
copper grid (200 mesh) and negatively stained using phospho-
tungstic acid (1% w/v) with the excess stain removed using a
filter paper. The grid was completely dried at ambient tem-
perature and the measurements were performed with a TEM
microscope (JEM-1230, Jeol, Tokyo, Japan).
Particle Size The particle size of the cubosomal disper-
sions were determined by using photon correlation spectros-
copy. Samples were diluted (100-fold) with de-ionized water
and placed in a scattering chamber where the light scattering
was monitored at 90° scattering angle using a Zetasizer Nano
ZS (Malvern Instruments Ltd., Worcestershire, U.K.). All
measurements were performed at 25±0.5°C in triplicate.
%EE CB loaded cubosomes were separated from unen-
trapped drug by centrifuging 2 mL of CB cubosomal disper-
sion in eppendorf tube at 12000 rpm for 30 min at 4°C (cen-
trifuge model 5417R, eppendorf, Hamburg, Germany). The
supernatant was diluted with the mobile phase and the free
unentrapped CB concentration in the resulting solution was
assayed by HPLC method mentioned earlier. The entrapment
efficiency was calculated using the following equation26)
Total CB in dispersion
Free unentrapoed CB in dispersion
(%EE) 100
Total CB in dispersion
−
=×
(6)
In Vitro Drug Release Studies The in vitro release of CB
from the cubosome dispersion was determined using United
States Phar macopeia (USP) dissolution tester, apparatus II
(Erweka, Germany). Cubosome dispersion containing the
equivalent of 97.8 mg CB was placed in a Spectra/Por® di-
alysis membrane of 12000 –14000 molecular weight cutoff and
sealed. The membrane was washed with distilled water several
times to remove preservative and soaked in release medium
overnight before use. The dispersion filled membrane was
introduced into the dissolution apparatus cups using 1000 mL
phosphate buffer pH 6.8 containing 1% SLS to maintain sink
conditions due to poor solubility of CB in phosphate buffer.27)
The release study was carried out at 37±0.5°C, and the stir-
ring shafts were rotated at a speed of 100 rpm. Three milliliter
samples were withdrawn periodically at predetermined time
intervals of 5, 10, 15, 30, 45 and 60 min and replaced instantly
by equal amount of fresh release medium to maintain a con-
stant volume. Samples were filtered using 0.22 µm filter and
analyzed by HPLC method previously described. All release
experiments were done in triplicates.
The release of the optimized CB cubosome dispersion and
free CB powder were compared using a buffer transition re-
lease media to assess the effect of changing pH conditions
on CB release through its movement along human GIT. The
release study was done in a USP type II apparatus rotated at
100 rpm at a temperature of 37±0.5°C. The acid stage com-
prised the use of 750 mL 0.1
N HCl buffer as a release me-
dium for 2 h, after which the pH was adjusted to pH 6.8 with
250 mL 0.2 M sodium triphosphate buffer21,28,29) and the experi-
ment was continued for 6 h. One percent SLS was added to
achieve sink conditions in pH 6.8. Samples were withdrawn
at 0.25, 0.5, 0.5,1, 1.5, 2, 2.5, 3, 4, 6 and 8 h, filtered and
analyzed using the same method described earlier The release
profiles were compared using similarity factor (f2) defined by
the following equation30)
0.5
tt
1
1
f 2 50log 1 100
n
t
RT
n
−



−







=
=+ ×
(7)
Where, n=the number of sampling time points, Rt=mean %
released of the reference at a given time point t, Tt=mean %
released of the test at the same time point t. The similarity
factor fits the result between 0 and 100. It is 100 when the two
release profiles are identical.31) In order to consider similar
release profiles, f2 values should be higher than 50, whereas
smaller values imply an increase in the dissimilarity between
release profiles over all time points.
In Vivo BT The blood clotting properties are considered
as an indicator for the extent of absorption of CB from the
optimized cubosomal dispersion compared to the commercial
marketed Plavix® tablet using a parallel randomized design.
The protocol of the study was reviewed and approved by the
Research Ethics Committee (protocol serial number PI 1387)
at Faculty of Pharmacy, Cairo University (Cairo, Egypt) and
the rabbit cuticle BT model described by Wong et al.32) was
used. Eighteen male New Zealand white rabbits weighing
2.5±0.11 kg each were randomly distributed between three
groups each of six rabbits. The rabbits were housed individu-
ally in stainless steel cages and fed a commercial diet. In a
dose of 20 mg/kg, group 1 received the optimized formula
CL1 while group 2 was administered the marketed product
(Plavix®, Sanofi Aventis, Cairo, Egypt) by oral gavage with
1 mL water.33) Group 3 served as a control and did not receive
any treatments. Two hours after the administration, the ani-
mals were anesthetized with ketamine (50 mg/kg+50 mg/kg/h
Intra-muscular (IM)) and xylazine (10 mg/kg+10 mg/kg/ h

1168 Vol. 66, No. 12 (2018)
Chem. Pharm. Bull.
IM).32) The anesthetized rabbits hind paws were shaved and a
standard cut was made at the apex of the cuticle with a razor
blade. Blood was allowed to flow freely by keeping the bleed-
ing site in contact with 37°C lactated Ringer’s solution. BT,
which is defined as the time after transection when bleeding
ceased was measured and the average was calculated.
Statistical Analysis The data obtained from different
tests were analyzed for statistical significance by one-way
ANOVA using Minitab® software (version 17, Minitab Inc.,
U.S.A.) followed by post hoc multiple comparisons using the
least square difference (LSD). Differences were considered to
be significant at p≤0.05.
Results and Discussion
Determination of CB Solubility in Different pH Media
Solubility studies were done to confirm the poor solubility
of CB in intestinal pH conditions and to select the appropri-
ate release medium in this study. As shown in Table 2, CB is
freely soluble in lower pH values while practically insoluble
in water and higher pH which comes in agreement with previ-
ous studies.27) A direct relationship is evident between pH and
solubility of CB, with solubility decreasing as the pH increas-
es which confirms the poor solubility of CB in intestinal pH
(6.8) and accounts for its poor bioavailability. Addition of 1%
SLS to phosphate buffer (pH 6.8) resulted in marked increase
in CB solubility in this buffer, thereby would be added to the
release medium in this study to achieve sink conditions.
Preparation of CB Loaded Cubosome Dispersions Ac-
cording to Table 1, twenty seven clopidogrel loaded cubosom-
al dispersions were successfully prepared (CL1 to CL27). All
formulas gave transparent gel prior to the addition of water.
After the addition of water they gave milky dispersions. GMO
was used in the preparation of cubosomes in this study due to
its ability to spontaneously form cubic phases upon mixing it
with water,34) in addition to being a safe, non-toxic, biocom-
patible and biodegradable ester.16) PL407 and PVA were used
as a surfactant and stabilizer respectively.22)
TEM The morphology of the cubosome dispersion inves-
tigated using TEM in Fig. 1 clearly confirms the formation of
cubosome nanoparticles. Micrographs also demonstrate that
the prepared cubosomes are in the nano range, non-aggregated
and well separated from each other.
Particle Size The particle size calculated from the TEM
images is comparable and in good agreement with that mea-
sured by the Zetasizer (Table 3) ensuring that all the prepared
formulae successfully yielded cubosome particles in the nano
range between 115±6.47 to 248±4.63 nm.
By examining the main effects of factors tested on particle
size, it is clear from Fig. 2B that the par ticle size is mostly
affected by the ratio of the drug to the disperse phase with
significant increase (p<0.05) in particle size when the ratio
increases (Fig. 2A). This finding comes in line with previ-
ous reports.13,35) On the contrary, it is clear from Fig. 2A,
that increasing PL407 concentration significantly ( p<0.05)
decreased the particle size. This can be attributed to the abil-
ity of PL407 as a surfactant to decrease the surface tension
and consequently decrease the surface energy of the cubosome
nanoparticles, thereby preventing particle aggregation36) and
decreasing particle size.37) These findings also endorse PL407
in cubosome formulations as the main responsible ingredient
for its stability.38) Interestingly it was observed that increas-
ing PVA concentration had insignificant effect on particle size
(p>0.05). Significant interactions were absent between factors
tested as shown in Fig. 2C confir med by two way ANOVA
(p>0.05)
%EE All the prepared cubosome nanoparticles achieved
successfully entrapped CB with high %EE ranging from
91.2 2±4.09 to 98.98±3.21% (Table 3). This success can be
attributed to the lipophilic nature of CB39)which causes it to
possess high affinity to the hydrophobic region of the cubic
phase.13)
As evident from the main effects plot (Fig. 3A) and pareto
plot (Fig. 3B), the %EE was only affected by the drug to the
disperse phase ratio where the %EE significantly ( p<0.05)
decreased as the ratio of the drug to the disperse phase was
increased. This pattern could be attributed to the saturation of
the bulk cubic phase with the drug due to its lipophilic nature,
thereby causing a disturbance of the bulk cubic phase mak-
ing the dr ug escape to the aqueous medium.13) On the con-
trar y, both PL407 and PVA concentrations had insignificant
(p>0.05) effects on %EE. No significant interactions were
detected between PL407 concentration and PVA concentra-
tion or PVA concentration and ratio of drug to disperse phase
(p>0.05) as shown in Fig. 3C which was confirmed by two
way ANOVA. However, a significant interaction was found
between PL407 concentration and the ratio of drug to disperse
phase confir med by two way ANOVA ( p<0.05). This interac-
tion can be explained by the effect of PL407 as a surfactant
on the partitioning and the solubilization of CB in the hydro-
phobic region of the cubosome thus affecting its entrapment
inside cubosome nanoparticles.4 0)
In Vitro Drug Release Studies As depicted from Figs.
4A–C, all the prepared CB cubosome dispersions exhibited
a biphasic release behavior in phosphate buffer containing
1%SLS with a rapid burst release after only 15 min (% Q15 ca.
be twee n 58.71±7.23 to 93.21±5.22%) followed by a slower
rate of release. This profile agrees with the previous reports
Table 2. Solubility of Clopidogrel Bisulphate in Different pH Media
Solvent Solubility (mg/L)
HCl Buffer (pH 1.2) 594.23±4.23
HCl buffer (pH 2.0) 543.72±14.46
Phosphate buffer (pH 5.5) 21.75±7.56
Phosphate buffer (pH 6.8) 19.5±4.53
Phosphate buffer (7.4) 15.4±2.42
Phosphate buffer (pH 6.8) with 1% SLS 408.23±24.23 Fig. 1. TEM Micrograph of CB Cubosome Dispersion

Vol. 66, No. 12 (2018) 1169
Chem. Pharm. Bull.
by Boyd and Nasr who studied the release of lipophilic dr ugs
from cubosome particles.12, 41) The burst release pattern could
be explained by the ability of the cubosome nanoparticles to
keep the insoluble CB in a solubilized nano size state with
the formation of concomitant large surface area for the diffu-
sion of the drug from the nanopar ticles upon exposure to the
release medium in appropriate sink conditions.20,42 ,43) While
the tortuosity and the narrow pore size of the inner aqueous
Table 3. The Measured Responses of Different Prepared CB Loaded Cubosome Dispersions and the Resultant Desirability Index
Formulae Particle size±S.D. (nm, n=3) %EE±S.D. (n=3) %Q15±S.D. (n=6) Desirability index
CL1 153±4.35 98.98±3.21 93.21±5.22 0.867668
CL2 139±7.73 96.21±5.89 71.15±6.47 0.700690
CL3 165±5.84 97.21±9.46 70.49±5.77 0.759330
CL4 143±8.27 95.18±2.47 77.64±4.12 0.620976
CL5 138±8.46 94.76±3.46 71.42±5.77 0.725396
CL6 148±4.26 96.43±7.41 74.13±2.31 0.616462
CL7 124±9.25 97.21±2.76 63.08±2.12 0.534747
CL8 115±6.47 95.65±5.47 78.41±5.47 0.593242
CL9 138±9.34 96.21±8.47 61.39±6.77 0.504888
CL10 169±5.46 97.29±9.42 73.95±2.84 0.718047
CL11 149±9.47 93.34±4.95 82.98±7.51 0.605815
CL12 185±4.47 95.42±9.46 72.17±3.43 0.590942
CL13 149±5.85 97.32±7.83 79.71±6.78 0.638576
CL14 148±6.46 97.21±1.47 89.27±3.58 0.840669
CL15 179±4.75 96.39±5.76 78.32±8.39 0.621149
CL16 136±4.57 96.52±2.34 64.34±1.92 0.468708
CL17 169±4.46 97.93±4.58 68.56±2.85 0.556770
CL18 149±5.35 97.12±6.46 62.56±3.09 0.435011
CL19 165±6.47 97.47±7.49 85.88±1.89 0.812696
CL20 195±5.46 95.19±3.68 68.64±5.24 0.370924
CL21 186±7.46 95.07±2.31 67.86±9.53 0.199958
CL22 243±4.55 91.56±5.67 75.05±2.08 0.223551
CL23 238±6.47 94.73±3.33 88.91±3.81 0.312010
CL24 248±4.63 92.21±4.69 70.48±5.86 0.193021
CL25 194±2.46 93.19±2.92 60.41±3.04 0.155169
CL26 178±3.85 91.22±4.09 64.43±6.67 0.183717
CL27 188±4.56 93.21±5.61 58.71±7.23 0.121166
Fig. 2. Main Effect Plot, Intera ction Plot and Pareto Plot of PL407 Concentr ation, PVA Concentration and Drug to Disperse Phase Ratio on Particle
Size for CB Loaded Cubosome Dispersions

1170 Vol. 66, No. 12 (2018)
Chem. Pharm. Bull.
nanochannels of the cubic nanoparticles were responsible and
contributed to the slowing down of the release in the second
phase.4 4,45) This pattern can be highly advantageous for the
treatment of patients with high risk of myocardial infarction
and stroke as the initial rapid drug release phase is important
to achieve high CB concentration in a very short time, while
the slow steady state release of the remaining product would
provide successful drug delivery along the time. Concurrently,
the initial burst release of the dr ug will be well tolerated as
higher doses (up to 600 mg) of CB were previously reported
in literature and were well tolerated with no adverse effects.46)
Buffer transition release study was used to assess optimized
cubosome dispersion CL1 compared to pure CB to mimic
the gastrointestinal conditions and to assess the release of
the drug along stomach and intestinal pH where it will be
absorbed. According to previous studies which concluded
maximum gastric emptying time of 2 h47) and maximum small
intestine transit time 6 h,48) at which the formula is expected
to fully release the drug.49) Therefore, the release study was
conducted for 2 h in gastric pH (1.2) and for 6 h in intestinal
pH (6.8).
As evident in Fig. 5, CB % released in both formulas
has reached 100% in gastric pH as CB is freely soluble in
acidic medium. This is expected as CB is a week base with
(pKa=4.5), which causes its ionization in lower pH39) however,
it will subsequently hinder its absor ption.6) Upon increas-
ing pH to 6.8 to mimic the intestinal pH, % released of CB
significantly (p<0.05) dropped to 66.82±4.12% after 6 h due
to formation of unionized free base which have poor solubil-
it y.50) On the contrary, optimized cubosome dispersion CL1
preserved %released of CB at 95.66±1.87 % ( p>0.05) which
can be attributed to the high affinity of CB to the lipid layer
of cubosomes due to its hydrophobic nature39) thereby retain-
ing CB in solubilized state13,19) and preventing its precipitation.
These findings are further endorsed by similarity factor higher
lower than 50 (f2=14) which confir m the success of cubosome
dispersion to enhance the in vitro release of CB in simulated
GIT conditions and prevent CB precipitation in the intestinal
pH.
By inspection of the main effects plot (Fig. 6A) of various
variables on drug release (%Q15), it is observed that increasing
PVA concentration insignificantly affected %Q15. On the other
hand increasing the ratio of dr ug to disperse phase signifi-
cantly decreased %Q15. This was expected if one considered
the increase in particle size discussed earlier which resulted
in a decrease in the surface area available for drug release.37)
Nevertheless, the concentration of PL407 had the most sig-
nificant (p<0.05) effect on the %Q15 as shown in Pareto plot
in Fig. 6B with %Q15 decreases as PL407 increases. This can
be ascribed to the coexistence of both Pn3m (CD) structure in
which PL407 molecules are mostly absorbed on the surface
with few PL407 forming the internal region of the cubosomal
particles and Im3m (CP) structure in which the excess PL407
was incorporated with GMO in the inner core after the satu-
ration of the cubosomes surface. As the PL407 increases, a
phase transition takes place from CD to CP with restricted
and lower drug release efficiency than CD.38,51,52) Further-
more, the two way ANOVA shows insignificant interactions
(p>0.05) between the tested factors as shown in Fig. 6C.
Based on the optimization of the factorial design and the
desirability index (Table 3), formula CL1 showed the highest
desirability index (D=0.867) among the other formulas and
the best attributes of high drug content and highest %Q15 of
CB release in intestinal pH (Fig. 4A). This can be ascribed to
its low ratio of drug to disperse phase (1 : 10) and low concen-
tration of PL407 (2.5%) which proved through the analysis of
the factorial design to be favourable formulation aspects for
CB cubosomal dispersions. On the contrary, CL27 exhibited
Fig. 3. Main Effect Plot, Interaction Plot and Pareto Plot of PL407 Concentration, PVA Concentration and Drug to Disperse Phase Ratio on %EE for
CB Loaded Cubosome Dispersions

Vol. 66, No. 12 (2018) 1171
Chem. Pharm. Bull.
the lowest desirability (D=0.121) and the lowest CB release
(%Q15=58.71±7.23) as shown in Fig. 4C. These results were
consistent with CL27composition of high ratio of drug to
disperse phase (3 : 10) and high PL407 concentration (10%)
as well which proved to be unfavourable for CB loaded cubo-
somes formulation according to the statistical design. Thus,
CL1 was further progressed to in vivo clinical assessment of
its antihaemostatic effect through bleeding time measurement.
In Vivo Bleeding Time CB, being a BCS class II drug,
its poor solubility in the intestine is a limiting step in its ab-
sorption in vivo. Thus, increasing its release in intestinal pH
in vitro will probably lead to enhanced drug levels in blood
stream.2 7,53) However, enhanced CB intestinal release is not
adequate to prove the potential success of the prepared cubo-
somal dispersion in vivo. Thus assessing the effect on blood
clotting and haemostasis of the prepared formula in vivo is of
equal if not much importance for consideration when assess-
ing cubosomal dispersions. Bleeding time has been a mainstay
Fig. 4. Drug Release Profiles of CB Loaded Cubosome Dispersion in Phosphate Buffer pH 6.8 Containi ng 1% SLS at 37±0.5°C (n=6) for (A) CL1 to
CL9, (B) CL10–CL18 and (C) CL19–CL27 and Free Drug
(Color fig ure ca n be accessed in the online version.)
Fig. 5. Dr ug Release Profiles of CB Optimized Cubosome Dispersion
CL1 and Free CB Powder in Buf fer Transition System (pH 1.2 for 2 h
Then pH 6.8 Cont aining 1% SLS for 6 h) at 37±0.5°C (n=6)

1172 Vol. 66, No. 12 (2018)
Chem. Pharm. Bull.
clinical assessment approach for measuring the haemostasis
and blood clotting behaviour which was endorsed as a direct
reflection technique to the in vivo effectiveness and the ad-
equacy of the measurement to the antithrombotic activity.32)
It is evident from Fig. 7 that cubosome dispersion CL1 had
significantly (p<0.05) increased BT (628.47±6.12 s) compared
to Plavix® (412.43±7.97 s) thereby confirming the increased
antihaemostatic action of cubosome nanoparticles compared
to commercial market. Both CL1 and Plavix® treatments
were significantly (p<0.05) different from the control group
(20 2.12±4.21 s) thus ensuring that the recorded differences
CL1 significantly increased BT compared to Plavix® can be
directly attributed to the increased CB levels in blood stream,
which in turn can be directly correlated to the enhanced CB
release and its rapid release and absorption in the intestine
mediated by the developed cubosome nanoparticles.13,32,54)
This can be ascribed to the digestion of GMO lipid by the
pancreatic lipases to form mixed micelles, cubic, hexagonal
and vesicular phases.13,20) At this environment, the drug re-
leased into the gastrointestinal fluid will be solubilized in the
micelles or mixed micellar structures thereby preventing pre-
cipitation in the intestinal pH and will be ready for immediate
absorption.15)
Based on the evidence in this study, cubosome nanopar-
ticles offer a promising technique for increasing CB solubility
in intestinal pH, the main site of its absorption with excel-
lent entrapment efficiency and adequate particle size. The
current work demonstrated that the enhanced release of CB
cubosomal dispersion has indeed associated with an in vivo
enhanced blood flow compared to marketed product Plavix®
in rabbits, thereby endorsing the merits of cubosomes as a
promising drug delivery system for CB. The factorial design
applied in this study demonstrated that the ratio of the drug
to the disperse is the most significant and contributing factor
affecting the attributes of the prepared cubosome nanopar-
ticles followed by PL407 concentration making these factors
a critical decision in the formulations of these systems. Unlike
PVA concentration which proved to be insignificant during
cubosome nanoparticles formulations. These findings pave the
road for cubosome nanoparticles to offer a new platform for
enhancing the release properties of BCS class II drugs and
improving their in vivo performance. Further research in this
area is recommended on different drugs to explore the cubo-
somes potential to enhance oral bioavailability of BCS class II
drugs in vitro and in vivo.
Conflict of Interest The authors declare no conflict of
interest.
Fig. 6. Mai n Effect Plot, Interaction Plot and Pareto Plot of PL407 Concent ration, PVA Concentration and Drug to Disperse Phase Ratio on %Q15 for
CB Loaded Cubosome Dispersions
Fig. 7. Histogram Comparing BT of Optimized CB Loaded Cubosomal
Disper sion CL1, Plavix® and Control in Rabbits (n=6)

Vol. 66, No. 12 (2018) 1173
Chem. Pharm. Bull.
References
1) Kim Y. I., Kim K. S., Suh K. H., Sha nmugam S., Woo J. S., Yong
C. S., Choi H. G., Int. J. Pharm., 415, 129–139 (2011).
2) Sangkuhl K., Klein T. E., Altman R. B., Pharmacogenet. Genomics,
20, 463– 465 (2010).
3) Tan C., Degim I. T., Pharm. De v. Technol., 17, 242–250 (2012).
4)
Savi P., Herber t J., Pflieger A., Dol F., Delebassee D., Combalbert J.,
Defrey n G., Maffrand J., Biochem. Pharmacol., 44, 527–532 (1992).
5) Farid N. A., Kurihara A., Wrighton S. A., J. Clin . Pharmacol., 50,
126 –14 2 (2 010).
6) Bali D. E., Osman M. A., El Maghraby G. M., Eur. J. Drug Metab.
Pharmacokinet., 41, 807–818 (2016).
7) Lassoued M. A., Sfar S., Bouraoui A., Khemiss F., J. Pharm. Phar-
macol., 64, 541–552 (2012).
8) Kara źniewicz-Łada M., Danielak D., Burcha rdt P., Kruszyna Ł.,
Komosa A., Lesiak M., Glówka F., Clin. Pharmacokinet., 53, 155–
164 (2014 ).
9) Wgih M. P., Patel J. S., Int. J. Pharm. Pharm. Sci., 2, 12–19 (2010).
10) Pouton C. W., Porter C., Adv. Dr ug Deliv. Rev., 60, 625– 637 (2008).
11) Hart nett T. E., O’Connor A. J., Ladewig K., Expert Opin. Drug
Deli v., 9, 1–15 (2015).
12) Boyd B., Int. J. Pharm., 260, 239–247 (2003).
13) Lai J., Chen J., Lu Y., Sun J., Hu F., Yin Z., Wu W., A APS
Ph ar mS ciTech , 10, 960–966 (2009).
14) Tayel S. A., El-Nabarawi M. A., Tadros M. I., Abd-Elsalam W. H.,
Dru g Deliv., 23, 3266 –3278 (2016).
15) Yang Z., Chen M., Yang M., Chen J., Fang W., Xu P., Int. J. Nano-
medicine, 9, 327–336 (2014).
16) Ganem-Quintanar A., Quintanar-Guerrero D., Buri P., Drug Dev.
Ind. Ph arm., 26, 809–820 (2000).
17) Kwon T. K., Hong S. K., Kim J.-C., J. Ind. Eng. Chem., 18, 563–567
(2012).
18) Yang Z., Tan Y., Chen M., Dian L., Shan Z., Peng X., Wu C., AAPS
Ph ar mS ciTech , 13, 1483 –1491 (2012).
19) Ali M. A., Kataoka N., Ran neh A. H., Iwao Y., Noguchi S., Oka T.,
Itai S., Chem. Pharm. Bull., 65, 42–48 (2017).
20) Nasr M., Dawoud M., J. Dr ug Deliv. Sci. Technol., 35, 106–113
(2016).
21) “The United States pharmacopeia 39/National For mulary 34,” The
United States Pharmacopeial Convention Inc., Rockville, MD, 2016.
22) Morsi N. M., Abdelbar y G. A., Ahmed M. A., Eur. J. Pharm. Bio-
pharm., 86, 178–189 (2014).
23) Abdelwahed N. A. M., Ahmed E. F., El-Gammal E. W., Hawas U.
W., 3 Biotech., 4, 533–544 (2014).
24) Elkomy M. H., El Menshawe S. F., Eid H. M., Ali A. M., Drug Dev.
Ind. Ph arm., 43, 531–544 (2017).
25) Der ringer G., Suich R ., J. Qual. Technol., 12, 214 –219 (1980) .
26) Thapa R. K., Baskara n R., Madheswaran T., Kim J. O., Yong C. S.,
Yoo B. K., J. Drug Deliv. Sci. Technol., 22, 479– 484 (2012).
27) Jassim Z. E., Hussein A. A., Int . J. Pharm. Pharm . Sci., 6, 838–851
(2014).
28) Lee Y. S., Song J. G., Lee S. H., Han H. K., Drug De liv., 24, 1731–
1739 (2 017) .
29) Feng D., Peng T., Huang Z., Singh V., Shi Y., Wen T., Lu M., Quan
G., Pan X., Wu C., Pharmaceutics, 10, E53 (2018).
30) Javadzadeh Y., Shariati H., Movahhed-Danesh E., Nokhodchi A.,
Drug Dev. Ind. Pharm., 35, 243–251 (2009).
31) Moore J. W., Flanner H. H., Pharm. Technol., 20, 64–74 (1996).
32) Wong P. C., Crain E. J., Watson C. A., Jiang X., Hua J., Bostwick J.
S., Martin L., J. Cardiovasc. Pharmacol., 49, 316–324 (2007).
33) Molero L., Lopez-Far re A., Mateos- Caceres P. J., Fernandez-San-
chez R., Luisa Maestro M., Silva J., Rodriguez E., Macaya C., Br. J.
Pharmacol., 146 , 419–424 (2005).
34) Shah J. C., Sadhale Y., Chiluk uri D. M., Adv. Drug Deliv. Rev., 47,
229–250 (20 01).
35) Esposito E., Cortesi R., Drechsler M., Paccamiccio L., Mariani P.,
Contado C., Stellin E., Menegat ti E., Bonina F., Puglia C., Pharm.
Res., 22, 2163–2173 (2 005).
36) Bar nard A. S., Zapol P., J. Chem. Phys., 121, 4276– 4283 (200 4).
37) Dora C. P., Singh S. K., Sanjeev Kumar A., Datusalia K., Deep A.,
Acta Pol. Pharm., 67, 283–290 (2010).
38) Esposito E., Eblovi N., Rasi S., Drechsler M., Gregor io G. M. D.,
Menegatt i E., Cortesi R., AA PS P ha rm Sc iTech, 5, 1–15 (2003).
39) Remko M., Remkova A., Broer R., Int. J. Mol. Sci., 17, 388 (2016).
40) Sharma N., Madan P., Lin S., Asian J. Pharm. Sci., 11, 404 –416
(2016).
41) Nasr M., Ghorab M. K., Abdelazem A., Acta Pharm Sin . B, 5,
79–88 (2015).
42) Swain S., Patra C. N., Rao M. E. B., “Pharmaceutical Drug Deliv-
ery Systems and Vehicles,” Woodhead Publishing Ltd., I ndia, 2016.
43) Magenheim B., Levy M. Y., Benita S., Int. J. Pharm., 94, 115–123
(1993).
44) Clogston J., Caffrey M., J. Control. Release, 107, 9 7–111 (2 00 5) .
45) Boyd B. J., Whitt aker D. V., Khoo S. M., Davey G., Int. J. Pharm.,
309, 218–226 (2006).
46) Herber t J. M., Frehel D., Vallee E., Kieffer G., Gouy D., Berger Y.,
Necciari J., Defreyn G., Maffrand J. P., Cardiovasc. Dr ug Rev., 11,
180–198 (1993).
47) Vasavid P., Chaiwatanarat T., Pusuwan P., Sr itara C., Roysri K.,
Namwongprom S., Kuanrakcharoen P., Premprabha T., Chunlertrith
K., Thongsawat S., Sirinthor npunya S., Ovartlarnporn B., Kachi n-
torn U., Leela kusolvong S., Kositchaiwat C., Chakkaphak S., Gon-
lachanvit S., Neurogastroenterol. Motil., 20, 371–378 (2014).
48) Davis S. S., Hardy J. G., Fara J. W., Gut, 27, 886–892 (1986).
49) Rawat M., Saraf S., Saraf S., AA PS Pha rmSciTe ch , 8, 289–297
(2007).
50) Zupancic V., Smrkolj M., Benkic P., Simonic I., Plevnik M., R itlop
G., Kristl A., Vrecer F., Acta Chim. Slov., 57, 376–385 (2010).
51) Nakano M., Sugita A., Matsuoka H., Handa T., Langmuir, 17,
3917–3922 (2001).
52) Zhao Y., Zhang J., Zheng L. Q., Li D. H., J. Disper. Sci. Technol.,
25, 795–799 (20 04).
53) Papich M. G., Martinez M. N., AAPS J., 17, 948–964 (2015).
54) Hoffmann P., Bernat A., Savi P., Herbert J. M., J. Pharmacol. Exp.
The r., 286, 670 –675 (1998).