Carvone Schiff base of isoniazid as a novel antitumor agent: Nanoemulsion development and pharmacokinetic evaluation

Article abstract of DOI:10.1016/j.molliq.2014.12.037

In the present study, various nanoemulsion formulations of carvone Schiff base of isoniazid (CSB-INH) were developed by aqueous phase titration method in order to evaluate its anticancer potential. Developed nanoemulsions of CSB-INH were characterized in terms of thermodynamic stability, self-nanoemulsification efficiency, droplet size, polydispersity index (PI), zeta potential (ZP), viscosity, refractive index (RI), % transmittance (% T), surface morphology and in vitro drug release studies. Based on lowest droplet size (19.4 nm), least PI (0.189), lowest viscosity (29.6 cP), optimal values of ZP (- 30.8 mV) & RI (1.341), highest % T (98.9%), highest drug release profile (94.5% after 24 h) and the presence of lowest concentration of Triacetin (12% w/w), formulation N1 was selected for in vitro cytotoxicity and in vivo pharmacokinetic studies. Cytotoxicity studies on human colon cancer cells indicated that CSB-INH in optimized nanoemulsion is around nine times more efficacious than free CSB-INH. Pharmacokinetic studies in Albino rats showed rapid absorption (rate and extent) of CSB-INH from optimized nanoemulsion as compared to its suspension formulation. These results indicated the potential of developed nanoemulsion for oral delivery of CSB-INH for chemoprevention of colon cancer.

Full text of DOI:10.1016/j.molliq.2014.12.037

Journal of Molecular Liquids 203 (2015) 111–119
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Carvone Schiff base of isoniazid as a novel antitumor agent:
Nanoemulsion development and pharmacokinetic evaluation
Mashooq A. Bhat a, Muzaffar Iqbal a,b, Abdullah Al-Dhfyan c, Faiyaz Shakeel d,
⁎
a
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
Bioavailability Laboratory, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
b
c
Stem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital & Research Center, MBC-03, P.O. Box 3354, Riyadh 11211, Saudi Arabia
Center of Excellence in Biotechnology Research (CEBR), College of Science, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia
d
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study, various nanoemulsion formulations of carvone Schiff base of isoniazid (CSB-INH) were
developed by aqueous phase titration method in order to evaluate its anticancer potential. Developed
nanoemulsions of CSB-INH were characterized in terms of thermodynamic stability, self-nanoemulsification
efficiency, droplet size, polydispersity index (PI), zeta potential (ZP), viscosity, refractive index (RI), % transmit-
tance (% T), surface morphology and in vitro drug release studies. Based on lowest droplet size (19.4 nm), least
PI (0.189), lowest viscosity (29.6 cP), optimal values of ZP (−30.8 mV) & RI (1.341), highest % T (98.9%), highest
drug release profile (94.5% after 24 h) and the presence of lowest concentration of Triacetin (12% w/w), formu-
lation N1 was selected for in vitro cytotoxicity and in vivo pharmacokinetic studies. Cytotoxicity studies on
human colon cancer cells indicated that CSB-INH in optimized nanoemulsion is around nine times more effica-
cious than free CSB-INH. Pharmacokinetic studies in Albino rats showed rapid absorption (rate and extent) of
CSB-INH from optimized nanoemulsion as compared to its suspension formulation. These results indicated the
potential of developed nanoemulsion for oral delivery of CSB-INH for chemoprevention of colon cancer.
© 2014 Elsevier B.V. All rights reserved.
Received 16 December 2014
Received in revised form 25 December 2014
Accepted 26 December 2014
Available online 30 December 2014
Keywords:
Isoniazid schiff base
Nanoemulsion
Cytotoxicity
Droplet size
Pharmacokinetic
1. Introduction
to be most effective against Mycobacterium tuberculosis, hence it was se-
lected in the present investigation [6]. Recently, nanotechnology-based
Colon cancer has been reported as the third most common tumor
among various tumors [1]. In the recent years, the incidence of colon
cancer has been increased gradually, hence its treatment become a
focus of research around the globe [2–4]. Radiation therapy, surgery
and chemotherapy are the three commonly used modalities for the
cure and control of colon cancer [5]. In chemotherapy, the anticancer
drug 5-fluorouracil is the commonly used drug for the treatment of
colon cancer [1,5]. Recently, Bhat and Al-Omar (2013) synthesized
and investigated various terpene Schiff bases of anti-tubercular drug
isoniazid (INH) for the effective treatment of tuberculosis [6]. Among
various terpene Schiff bases investigated, the carvone Schiff base of
INH (CSB-INH) was found to be very potent and effective. Moreover,
various hydrazines, hydrazones, nicotinic acid and terpene derivatives
of INH have also been investigated successfully for the treatment of var-
ious cancers such as colon cancer, ovarian cancer, blood cancer, glioblas-
toma and tuberculosis [1,6–12]. Therefore, in the present study, CSB of
INH was synthesized, characterized and investigated for its anticancer
potential in the form of nanoemulsion. Previously, CSB-INH was found
drug delivery systems have been investigated effectively for the
targeting of various anticancer drugs to carcinoma cells in order to en-
hance therapeutic efficacy and to reduce the adverse effects of the treat-
ment both in vitro as well as in vivo [13–19]. Moreover, various
nanocarries have also been investigated as the effective drug delivery
vehicles for the targeting of INH and its analogues in order to reduce
the dose and adverse effects of the treatment [20–25]. Nanoemulsions
are oil/lipid based clear, transparent/isotropic and thermodynamically/
thermokinetically stable systems composed of oil/lipid, surfactant and
cosurfactant with droplet size in the range of 10–100 nm [13–15].
These novel nanovehicles have been investigated effectively for the
multipurpose drug delivery system in order to enhance in vitro dissolu-
tion rate/solubility, in vivo therapeutic efficacy and bioavailability of
various poorly water-soluble drugs [13–15,26–29]. Nanoemulsion
system offers several advantages over unstable dispersions (emulsions,
suspensions and colloids) [26]. These advantages include ease of
preparation, excellent solubilization capacity, physical stability, smaller
droplet size, lower viscosity and bioavailability enhancement [27,28].
CSB-INH is poorly water soluble compound (solubility: 0.11 mg/ml)
with log p value of 3.02 [6]. Till date, the antitumor potential of CSB-
INH via nanoemulsion approach has not been investigated in the litera-
ture so far. Therefore, in the present study, CSB-INH was synthesized
⁎
Corresponding author.
E-mail address: faiyazs@fastmail.fm (F. Shakeel).
http://dx.doi.org/10.1016/j.molliq.2014.12.037
0167-7322/© 2014 Elsevier B.V. All rights reserved.

112
M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
and characterized in order to investigate its antitumor effects against
human colon cancer cell lines.
cosurfactant, respectively for the development of nanoemulsions.
Deionized water selected as an aqueous phase. For the construction of
pseudo-ternary phase diagrams, Surfactant (Tween-80) and cosurfactant
(Transcutol-HP) were mixed in various mass ratios (1:0, 1:2, 1:1, 2:1 and
3:1). Triacetin (oil phase) and a particular mass ratio of Tween-80 to
Transcutol-HP (Smix) were mixed at mass ratios of 1:9 to 9:1. Pseudo-
ternary phase diagrams were developed by titrating the mixture of oil
phase and specific Smix slowly with deionized water (aqueous phase ti-
tration method) [28,29]. The physical state of nanoemulsions was
marked on a phase diagram with the first axis representing the aqueous
phase (water), second oil phase (Triacetin) and third representing
the specific mass ratio of surfactant (Tween-80) to cosurfactant
(Transcutol-HP).
2. Materials and methods
2.1. Materials
INH, carvone, polyoxy-35-castor oil (Cremophor-EL), ethanol and
polyoxyethylene (20) sorbitan monooleate (Tween-80) were pur-
chased from Sigma Aldrich (St. Louis, MO). Glycerol triacetate
(Triacetin) was purchased from Alpha Chemica (Mumbai, India). Pro-
pylene glycol monocaprylate-type II (Capryol-90), propylene glycol
monolaurate-type II (Lauroglycol-90), oleoyl macrogol-6-glyceride
(Labrafil-M1944CS), propylene glycol dicaprylocaprate (Labrafac-PG),
caprylo caproyl macrogol-8-glyceride (Labrasol) and diethylene glycol
monoethyl ether (Transcutol-HP) were kind gift samples from
Gattefossé (Lyon, France). Iso-octylphenoxypolyethoxyethanol
(Triton-X100), propylene glycol (PG) and polyethylene glycol-400
(PEG-400) were purchased from BDH Laboratories (Liverpool, UK).
Chromatographic grade acetonitrile was purchased from BDH Laborato-
ry (Liverpool, UK). HT-29 human colon cancer cell lines were purchased
from American type cell culture collection (ATCC, Manassas, VA). Ultra-
pure water was obtained in the laboratory from ELGA water purification
unit (Wycombe, Bucks, UK). All other solvents and reagents used were
of LR grade and obtained from E-Merck (Germany).
2.5. Formulation development
The maximum nanoemulsion zones were observed in 1:1 mass ratio
of Tween-80 and Transcutol-HP, hence this ratio was selected for the
preparation of CSB-INH nanoemulsions. From the pseudo-ternary
phase diagram, different nanoemulsions with formulation codes
of N1–N5 were selected (Table 1). Almost the entire range of
nanoemulsion zones in the phase diagram were taken into account
and varied Triacetin compositions (12, 16, 20, 24 and 28% w/w) with
minimum Tween-80 (12% w/w) and Transcutol-HP (12% w/w) concen-
tration were selected. 25 mg of CSB-INH was directly solubilized in each
nanoemulsion by vortexing at 1000 rpm and 25 1 °C for about 5 min.
After 5 min, the drug was completely solubilized in the system and CSB-
INH-loaded nanoemulsions were transferred into transparent glass vials
(Table 1).
2.2. Synthesis and characterization of carvove Schiff base of INH
CSB-INH was prepared by the reaction of carvone (0.15 g, 1 mmol)
with INH (0.14 g, 1 mmol) in ethanol/water (10 ml). Initially, INH was
dissolved in water and the alcoholic solution of carvone was added to
the INH solution. This mixture was stirred at room temperature for
about 1–3 h and then concentrated under reduced pressure. The residue
obtained was purified by washing with cold ethanol and ethyl ether.
Colorless blocks of compounds were prepared by recrystallization
with ethanol by the slow evaporation of solvent at room temperature.
The synthesized compound was characterized in terms of % yield, melt-
ing point, FTIR, 1H NMR, 13C NMR, mass spectra and elemental analysis
[6]. CSB-INH was synthesized according to Scheme 1. The molecular
structure of CSB-INH was confirmed by FTIR, 1H NMR, 13C NMR, mass
spectra and elemental analysis. This compound was successfully inves-
tigated as a potential anti-tubercular agent in previous studies [6]. In the
present study, its potential as a potent anti-tumor agent against human
colon cancer cell line was investigated.
2.6. Thermodynamic stability and self-nanoemulsification tests
Thermodynamic stability tests on prepared CSB-INH nanoemulsions
were carried out in order to remove unstable or metastable
nanoemulsions. These tests were performed in terms of centrifugation
(at 5000 rpm for 30 min), heating & cooling cycles (3 cycles between
4 and 50 °C for 48 h) and freeze–pump–thaw cycles (3 cycles between
−21 and +25 °C for 24 h) as reported in the literature [28,29]. The ob-
jective of the self-nanoemulsification efficiency test was to evaluate any
phase separation or precipitation upon dilution with water. In order to
perform this test, 1 ml of each CSB-INH nanoemulsion (N1–N5) was
diluted 500 times with deionized water. The efficiency of each
nanoemulsion was evaluated visually using the following A–E grading
systems as reported previously [26,29]:
Grade A: Rapid forming clear/translucent nanoemulsion
Grade B: Rapid forming bluish slightly less clear nanoemulsion
2.3. Screening of components for nanoemulsion preparation
Grade C: Slowly forming milky/turbid emulsions
Grade D: Dull, grayish slowly forming milky emulsions
Grade E: Emulsions with oil globules at the surface.
The selection of components in terms of oil phase, surfactant and co-
surfactant was based on the solubility of CSB-INH. Therefore, the satu-
rated solubility of CSB-INH in different oils (Triacetin, Capryol-90,
Lauroglycol-90, Labrafac-PG and Labrafil-M1944CS), different surfac-
tants (Tween-80, Labrasol, Cremophor-EL and Triton-X100), different
cosurfactants (Transcutol-HP, PEG-400, PG and ethanol) and water
was determined by adding the excess amount of CSB-INH in 2 ml of
each component in 5 ml capacity vials. All the mixtures were continu-
ously mixed in an isothermal water shaker bath (Julabo, MA) at
2.7. Physicochemical characterization of CSB-INH nanoemulsions
Prepared CSB-INH nanoemulsions were characterized physicochem-
ically in terms of droplet size distribution, polydispersity index (PI), zeta
potential (ZP), viscosity, refractive index (RI), percentage of transmit-
tance (% T) and transmission electron microscopy (TEM). The mean
droplet size, PI and ZP of CSB-INH nanoemulsions (N1–N5) were
recorded using Malvern Particle Size Analyzer/Zetasizer (Malvern
Instruments Ltd., Holtsville, NY) at room temperature at a scattering
angle of 90° as reported previously [30]. The viscosity and RI of CSB-
INH nanoemulsions (N1–N5) were recorded using Brookfield Viscome-
ter (Brookfield Engineering Laboratories, Middleboro, MA) and Abbes
type Refractometer (Precision Testing Instruments Laboratory,
Germany) at 25 1 °C, respectively as reported in the literature [28,
29]. The % T of CSB-INH nanoemulsions (N1–N5) was recorded
100 rpm and 37
0.5 °C for 72 h [26,28]. After 72 h, each mixture
was taken out from the shaker bath and centrifuged at 5000 rpm for
20 min, and the supernatant was diluted suitably with methanol and
subjected for analysis of CSB-INH content spectrophotometrically at
220 nm.
2.4. Construction of pseudo-ternary phase diagrams
Based on highest solubilization potential of CSB-INH, Triacetin,
Tween-80 and Transcutol-HP were used as oil phase, surfactant and

M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
113
Scheme 1. Synthesis of CSB-INH.
spectrophotometrically at the wavelength of 550 nm as reported previ-
ously [29].
TEM analysis (Tecnai TF20, Hillsboro, OR) was performed to evaluate
the surface morphology of droplets of optimized nanoemulsion N1. TEM
analysis was performed under light microscopy operating at 200 kV as
reported previously [26].
2.10. In vitro cytotoxicity studies
With the background to develop a suitable nanoemulsion formula-
tion to investigate the anticancer potential of CSB-INH, we investigated
in vitro therapeutic efficacy of optimized nanoemulsion N1 on HT-29
human colon cancer cell line. In vitro cytotoxicity (%) was evaluated by
exposing different molar concentrations of CSB-INH from optimized
nanoemulsion (N1) and free CSB-INH (aqueous solution). Optimized for-
mulation N1 without CSB-INH was also applied in the same volume as
applied for CSB-INH loaded N1 which was used as a control. A WST-1
[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetra-
zolium] assay was performed to assess the cytotoxic effects of CSB-INH
nanoemulsion (N1), CSB-INH solution and control. The HT-29 colon can-
cer cells were cultured in RPMI 1640 medium, supplemented with 10%
fetal bovine serum (FBS), 1% ABM (GIBCO), 2 mmol/l of L-glutamine,
100 IU/ml penicillin and 100 mg/ml streptomycin at 37 °C in a 5% CO2 at-
mosphere. Cells were seeded into 96-well plates at 0.4 × 104 cells/well
and incubated for 24 h in an incubator at 37 °C in a 5% CO2 atmosphere.
The medium was replaced with a fresh one containing the desired con-
centrations of aqueous solution, nanoemulsion (N1) and control in the
same volume. After 72 h, 10 μl of the WST-1 reagent was added to each
well and the plates were reincubated for 4 h at 37 °C. The amount of
formazan was quantified using ELISA reader at 450 nm.
2.8. In vitro drug release studies
In vitro drug release studies on CSB-INH nanoemulsions (N1–N5)
were performed to compare the release profile of CSB-INH from differ-
ent nanoemulsions and CSB-INH suspension, all having the same
amount of drug (25 mg). These studies were performed in 500 ml of
gastric buffer (pH 1.2) (dissolution media) using USP XXIV method at
100 rpm and 37 0.2 °C [26]. In order to perform these studies, 1 ml
each of CSB-INH nanoemulsion (N1–N5) and CSB-INH suspension was
placed in a ready-to-use dialysis bag. Three milliliters of samples was
withdrawn at regular time intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 18 and
24 h and the same amount of drug free fresh gastric buffer (pH 1.2)
was replaced every time. The samples were analyzed for CSB-INH
content spectrophotometrically at 220 nm.
2.9. Kinetic analysis of drug release data
The release mechanism of CSB-INH from developed nanoemulsions
was investigated using various kinetic models such as zero order,
first order, Higuchi, Hixson–Crowell and Korsemeyer–Peppas models
[31,32]. This analysis was performed using Microsoft excel 2010
program. The mathematical representation of these models is as
follows:
2.11. Pharmacokinetic study in rats
Twelve male Wistar albino rats weighing 210 15 g (mean SD)
were obtained from the Animal Care and Use Centre, College of Pharma-
cy, King Saud University, Riyadh, Saudi Arabia. Prior to the experiment,
the animals were acclimatized and kept in plastic cages under standard
laboratory conditions, a temperature of 25 2 °C and 55 5% RH with
a 12 h light/dark cycle and a pellet diet was given with water ad libitum.
All experiments were carried out according to the guidelines of the
Animal Care and Use Committee at King Saud University. In a single
dose parallel designed study, the rats were randomly divided into two
groups (6 in each) which served as CSN-INH suspension and CSB-INH
nanoemulsion (N1) treatment group, respectively. The rats were fasted
for at least 10 h before the experiments. Blood samples (approximately
0.75 ml) were collected from the retro-orbital plexus into heparinized
Zero order
Qt ¼ Q0 þ K0t
ð1Þ
ð2Þ
First order
K1t
2:303
LogC ¼ LogC0−
Higuchi
Qt ¼ kt0:5
ð3Þ
ð4Þ
Table 1
Composition of CSB-INH nanoemulsions (N1–N5) prepared using Triacetin, Tween-80,
Transcutol-HP and water.
Hixson–Crowell cube root
Code
Formulation composition (% w/w)a
Smix ratio
ꢀ
ꢁ
W301 −W3t1 ¼ kht
Triacetin
Tween-80
Transcutol-HP
Water
N1
N2
N3
N4
N5
12
16
20
24
28
12
12
12
12
12
12
12
12
12
12
64
60
56
52
48
1:1
1:1
1:1
1:1
1:1
Korsmeyer–Peppas
Qt
Q∞
¼ kptn:
ð5Þ
a
Twenty five milligrams of CSB-INH was incorporated into each nanoemulsion.

114
M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
microfuge tubes at predose and at 0.5 1, 2, 3, 4, 6, 8 and 24 h after admin-
istration of CSB-INH (5 mg/kg, oral) in both groups. Plasma samples
were harvested by centrifuging the blood at 45,00 ×g for 8 min and
stored frozen at −80 10 °C until analysis.
study, these components were screened based on highest solubility of
CSB-INH in these components. The saturated solubility data of CSB-INH
in different components at 37 °C is listed in Table 2. Among the different
oils investigated, the highest solubility of CSB-INH was observed in
Triacetin (42.42 3.84 mg/ml) followed by Lauroglycol-90, Capryol-90,
Labrafac-PG and Labrafil-M1944CS (Table 2). However, the highest solu-
2.12. Determination of CSB-INH concentration in plasma by UPLC MS/MS
bility of CSB-INH was observed in Tween-80 (67.24
6.13 mg/ml)
The ACQUITY™ UHPLC system coupled to a triple-quadruple tandem
mass spectrometer Micromass® Quattro micro™ (Waters Corp.,
Milford, MA, USA) was used to analyze the CSB-INH in rat plasma
samples using carbamazepine as an internal standard (IS). The
chromatographic separation was achieved on Acquity BEH™ C18 col-
umn (50 × 2.1 mm, I.D., 1.7 μm, Waters, USA) maintained at 40 °C.
The mobile phase was composed of acetonitrile-10 mM ammonium ac-
etate (95:5,v/v) at a flow rate of 0.3 ml/min. A triple-quadruple tandem
mass spectrometer equipped with electrospray ionization (ESI) inter-
face was used for the analytical detection of both analyte and IS. The de-
tection was performed in ESI positive mode using multiple reaction
monitoring (MRM) by the ion transitions of m/z 270.08 N 79.93 for
CSB-INH and m/z 237.0 N 178.97 for IS, having a dwell time of 0.106 s.
The optimized cone voltage and collision energy were 38 V and 32 eV
for CSB-INH and 32 V and 34 eV for IS, respectively. The collision gas
(argon) flow was 0.1 ml/min and capillary voltage was set at 3.5 kV.
The Mass Lynx software (Version 4.1, SCN 714) was used to control
the UHPLC-MS/MS system and data was collected and processed using
TargetLynx™ program. Samples were prepared by protein precipitation
method using acetonitrile (ACN). Plasma samples (200 μl) were trans-
ferred to 1.5 ml centrifuge tubes and 20 μl of IS (40 μg/ml in ACN) was
added. The samples were vortexed mixed for 20 s and 380 μl of ACN
was added. The samples were again vortex mixed gently for 1 min.
Samples were centrifuged at 12,500 ×g at 8 °C for 10 min. The superna-
tant was transferred to UPLC vials subjected to 5 μl for the analysis. The
proposed analytical method was validated according to the FDA and
EMEA guidelines [33].
among different surfactants followed by Cremophor-EL, Labrasol and
Triton-X100. On the other hand, the highest solubility was observed in
Transcutol-HP (76.84 7.86 mg/ml) among different cosurfactants in-
vestigated followed by ethanol, PEG-400 and PG. CSB-INH was found to
be poorly soluble in water (solubility 0.11 mg/ml) as shown in Table 2.
Based on the solubility data of CSB-INH, Triacetin, Tween-80 and
Transcutol-HP were selected as oil phase, surfactant and cosurfactant, re-
spectively. However, deionized water was selected as aqueous phase.
3.2. Nanophasic map construction and formulation development
Pseudo-ternary phase diagrams were constructed separately for
each Smix ratio and the results are presented in Fig. 1. From the phase di-
agrams, it was observed that Smix 1:0 showed relatively low
nanoemulsion zones (Fig. 1A). The highest amount of Triacetin (oil
phase) that was solubilized by this ratio was found to be 16% w/w
with a high percentage of Smix (64% w/w). However, in the case of
Smix 1:2 (Fig. 1B), when the cosurfactant (Transcutol-HP) was used
along with Tween-80 (surfactant), the nanoemulsion zones increased
rapidly as compared to the previous Smix ratio. The highest amount of
oil phase that was solubilized by 1:2 ratio was found to be 22% w/w
by incorporating 52% w/w of Smix. On the other hand, when Smix ratio
of 1:1 (Fig. 1C) was investigated, the nanoemulsion zones increased sig-
nificantly as compared to previous ratios (1:0 and 1:2 ratios). The
highest amount of oil phase that was solubilized by this ratio was
found to be 34% w/w by incorporating lower concentration of Smix
(30% w/w). These results showed the dominance of cosurfactant
(Transcutol-HP) along with surfactant (Tween-80) in nanosizing.
When Smix ratio of 2:1 was investigated (Fig. 1D), the nanoemulsion
zones started to decrease as compared to 1:1 ratio. The highest amount
of oil phase that was solubilized by 2:1 ratio was found to be 28% w/w
by incorporation of 41% w/w of Smix. When Smix ratio of 3:1 was inves-
tigated (Fig. 1E), the nanoemulsion zones decreased further as com-
pared to 1:1 and 2:1 ratios. The highest amount of oil phase that was
solubilized by 3:1 ratio was found to be 17% w/w by incorporation of
39% w/w of Smix. Phase titration studies showed that the highest
nanoemulsion zones were exposed by Smix ratio of 1:1 as shown in
Fig. 1C. Therefore, different nanoemulsion compositions were precisely
selected from Fig. 1C. Almost the entire nanoemulsion zones in Fig. 1C
were covered and different oil compositions (12, 16, 20, 24 and 28%
2.13. Pharmacokinetic calculation and data analysis
The plasma concentrations were used to construct pharmacokinetic
profiles by plotting drug concentration–time profile curves. All values
are expressed as the mean SD. To determine pharmacokinetic param-
eters, all obtained data were subsequently fed into PK software on
Microsoft excel®. The noncompartmental pharmacokinetic parameters
such as maximum plasma concentration (Cmax) and time to reach max-
imum concentration (Tmax), area under the curve from 0 to t (AUC0–24
)
and area under first moment curve from 0 to 24 (AUMC0–24), elimina-
tion rate constant (Kel) and half-life were calculated whereas, mean res-
idence time (MRT) is the ratio of AUMC0–24 to AUC0–24. Unpaired T-test
with two tail P value was used for statistical calculation using
GraphpadInstat software.
Table 2
Equilibrium solubility data of CSB-INH in various oils, surfactants and cosurfac-
tants at 37 °C (n = 3).
2.14. Statistical analysis
Sample matrices
Solubility SD (mg/ml)
In vitro drug release data, characterization parameters among CSB-
INH nanoemulsions (N1–N5) and in vitro cytotoxicity data were
statistically evaluated by one way analysis of variance (ANOVA)
using Dennett's test to evaluate the significant differences using
GraphpadInstat software. Data were considered statistically significant
at the value of p b 0.05.
Triacetin
42.42 3.84
15.24 1.38
18.62 2.08
8.34 0.81
7.13 0.65
67.24 6.13
32.15 2.65
37.26 3.76
25.81 2.21
76.84 7.86
28.61 2.52
19.23 1.78
61.23 5.34
0.11 0.00
Capryol-90
Lauroglycol-90
Labrafac-PG
Labrafil-M1944CS
Tween-80
Labrasol
Cremophor-EL
Triton-X100
Transcutol-HP
PEG-400
3. Results and discussion
3.1. Screening of components for nanoemulsion preparation
PG
Ethanol
Water
The saturated solubility of drug (CSB-INH) in oils, surfactants and co-
surfactants is the most important step for the screening of components
especially in oral drug delivery systems [26,28]. Hence, in the present
Polyethylene glycol-400 (PEG-400), propylene glycol (PG).

M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
115
Fig. 1. Pseudo-ternary phase diagrams with nanoemulsion zones (dotted area) for oil phase (Triacetin), aqueous phase (water), surfactant (Tween-80) and cosurfactant (Transcutol-HP) at
Smix ratios of A. 1:0, B. 1:2, C. 1:1, D. 2:1 and E. 3:1.
w/w) with fixed Smix concentration (24% w/w) were precisely selected.
25 mg of compound (CSB-INH) was directly encapsulated on each
nanoemulsion. The composition of CSB-INH loaded nanoemulsions is
listed in Table 1.
thermodynamic stability tests. Thermodynamically stable nanoemulsions
(N1–N5) were further evaluated for self-nanoemulsification efficiency.
The self-nanoemulsification efficiency test showed that formulations
N1–N4 passed this test with grade A while the formulation N5 passed
this test with grade B.
3.3. Thermodynamic stability and self-nanoemulsification tests
3.4. Physicochemical characterization of CSB-INH nanoemulsions
The objective of thermodynamic stability tests was to eliminate meta-
stable/unstable formulations. In order to develop robust and stable
nanoemulsions, developed formulations were subjected to different
The results of physicochemical parameters of CSB-INH nanoemulsions
are listed in Table 3. The mean droplet size of CSB-INH nanoemulsions

116
M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
(N1–N5) was observed in the range of 19.4–124.4 nm (Table 3). The
droplet size of prepared nanoemulsions was found to be reduced rapidly
by decreasing the concentration of oil phase (Triacetin) in the formula-
tions. The largest droplet size was observed in nanoemulsion N5
(124.4 14.8 nm) which was probably due to the presence of the highest
concentration of Triacetin (28% w/w) in N5. However, the droplet size of
nanoemulsion N1 was found to be lowest (19.4 2.3 nm) that could be
due to the presence of the lowest concentration of Triacetin (12% w/w) in
N1. The PI of CSB-INH nanoemulsions (N1–N5) was observed in the range
of 0.189–0.324 (Table 3). The PI of nanoemulsions N1–N3 was relatively
low (0.189–0.212), indicating good uniformity in droplet size as com-
pared to nanoemulsions N4 and N5. Nanoemulsion N1 showed the least
PI value (0.189), suggesting highest uniformity. However, the highest PI
value was observed in nanoemulsion N5 (0.324).
The ZP values of CSB-INH nanoemulsions (N1–N5) were observed in
the range of −42.6 to −30.8 mV (Table 3). The lowest value of ZP was
observed in nanoemulsion N5 (−42.6 mV). However, the highest one
was observed in nanoemulsion N1 (−30.8 mV). The negative net
charge on ZP for all CSB-INH nanoemulsions was possibly due to the
presence of fatty acid esters in Triacetin in all nanoemulsions as report-
ed previously in the literature [34].
was found to be rapid (immediate type). The difference in the release
profile of CSB-INH between nanoemulsions (N1–N5) and suspension
is highly significant (P b 0.05). More than 50% of CSB-INH was found
to be released from all nanoemulsions as compared to 15% from
suspension formulation after 8 h of study (Fig. 3). After 8 h, all
nanoemulsions and suspension formulation showed a slower release
of CSB-INH (sustained release profile). The highest drug release pro-
file of CSB-INH was observed in nanoemulsion N1 (Fig. 3). The cumu-
lative % amount of CSB-INH that was released from N1 after 24 h was
found to be 96.2% as compared to only 20.8% from suspension formu-
lation. More than 85% of CSB-INH was released from N1 after 8 h of
study. These results were in good agreement with the physicochem-
ical characterization of nanoemulsions. The highest release profile of
CSB-INH from N1 was possibly due to its lowest droplet size, lowest
viscosity, least PI, highest % T and the presence of lowest concentra-
tion of oil phase (Triacetin). Two steps release profile of CSB-INH
from developed nanoemulsions and suspension formulation indicat-
ed diffusion controlled dissolution rate of CSB-INH from all formula-
tions [28].
The viscosity of CSB-INH nanoemulsions (N1–N5) was observed in
the range of 29.6–72.3 cP (Table 3). The viscosity of prepared
nanoemulsions was also found to be reduced rapidly by decreasing
the concentration of oil phase in nanoemulsions. The lowest viscosity
was observed in nanoemulsion N1 (29.6 2.7 cP) which was probably
due to its lowest droplet size. However, the highest viscosity was ob-
3.6. Kinetic analysis of drug release data
In order to evaluate the accurate drug release mechanism, zero
order, first order, Higuchi and Hixson–Crowel models were selected. Be-
cause, the drug release was significant for up to 8 h from all formula-
tions, kinetic analysis was performed for up to 8 h drug release data.
The results of release kinetic parameters are listed in Table 4. The release
from all nanoemulsions (N1–N5) and suspension formulation followed
the Peppas model with non-Fickian diffusion mechanism (Table 4). Ac-
cording to the Peppas model, If the value of n is equal to 0.5, the release
mechanism is considered as diffusion controlled drug release (Fickian
mechanism). However, if the value of n is greater than 0.5 but less
than 1.0, the release mechanism is considered as non-Fickian diffusion
mechanism. On the other hand, the value of n greater than 1.0 is related
with supercase II transport mechanism [31]. The value of n in developed
nanoemulsions (N1–N5) and suspension formulation was observed in
the range of 0.877–0.982, indicating non-Fickian diffusion mechanism
in all formulations. Our results of kinetic analysis were in good
agreement with previous literature [35,36]. Based on lowest droplet
size, least PI, lowest viscosity, optimal values of ZP & RI, highest % T,
highest drug release profile and the presence of lowest concentration
of Triacetin, CSB-INH nanoemulsion N1 was selected for further
evaluation.
served in nanoemulsion N5 (72.3
7.4 cP) due to its largest droplet
size. The % T of CSB-INH nanoemulsions (N1–N5) was observed in the
range of 96.1–98.9% (Table 3). The highest % T was observed in
nanoemulsion N1 (98.9 0.4%). However, the lowest one was observed
in nanoemulsion N5 (96.1
0.4%). The results of % T measurements
showed the transparent nature of all nanoemulsions.
TEM analysis was performed to evaluate the surface morphology
and shape of droplets of optimized nanoemulsion N1. TEM Photomicro-
graphs of optimized nanoemulsion N1 were taken and interpreted
(Fig. 2). The size of all droplets of nanoemulsion N1 was found to be
less than 50 nm (Fig. 2). The shape of nanoemulsion droplets was
observed as non-spherical (ovoidal) which was probably due to the
presence of Triacetin and Tween-80 as reported by Anwer et al.
(2014) [34]. The droplet size distribution of optimized nanoemulsion
N1 recorded by TEM was in good agreement with the droplet size
distribution recorded by scattering technique (Malvern Particle Size
Analyzer).
3.5. In vitro drug release studies
In vitro drug release studies were performed via dialysis membrane
on CSB-INH nanoemulsions (N1–N5) and drug suspension in order to
get optimized nanoemulsion. The results of CSB-INH release from
nanoemulsions and drug suspension are presented in Fig. 3. The initial
release profile of CSB-INH from all nanoemulsions and drug suspension
Table 3
Physicochemical characterization of CSB-INH nanoemulsions (N1–N5).
Code Characterization parameters
Δdm SD (nm) PI
ZP (mV)
η
SD (cP) RI SD
% T SD
N1
N2
N3
N4
N5
19.4 2.3
43.8 4.6
57.3 6.3
104.2 9.7
124.4 14.8
0.189 −30.8
0.209 −31.9
0.212 −35.4
0.312 −40.3
0.324 −42.6
29.6 2.7
45.4 3.8
59.8 4.9
65.4 6.2
72.3 7.4
1.341 0.07 98.9 0.4
1.342 0.08 98.6 0.3
1.344 0.09 97.7 0.2
1.346 0.10 96.4 0.5
1.347 0.11 96.1 0.4
Mean droplet diameter (Δdm), polydispersity index (PI), viscosity (η), % transmittance (%
Fig. 2. Transmission electron microscopic (TEM) image of optimized CSB-INH
T), zeta potential (ZP), refractive index (RI), and standard deviation (SD).
nanoemulsion (N1) showing non-spherical droplets within submicrone range.

M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
117
Fig. 3. In vitro drug release profile of CSB-INH from different nanoemulsions (N1–N5) and
drug suspension via dialysis membrane.
Fig. 4. Cell viability of CSB-INH in aqueous solution (free CSB-INH), control and optimized
nanoemulsion N1 on HT-29 human colon cancer cell lines.
3.7. In vitro cytotoxic studies
Cell survival (%) was measured at 1.25, 2.5, 5, 10, 20, 40, 80 and
100 μM concentration of CSB-INH in an aqueous solution and N1
(Fig. 4). The highest cytotoxicity of free CSB-INH was observed as
45.60 4.32% at a molar concentration of 100 μM (Table 5). However,
the same molar concentration of CSB-INH in optimized nanoemulsion
more efficacious than free CSB-INH which could reduce the dose of
CSB-INH as well as its gastrointestinal adverse effects. Schiff bases
have been investigated as good ligands for metals, hence the purported
mechanism of action of CSB-INH could possibly be based on the forma-
tion of metal complexes which are known to inactivate the enzymes re-
sponsible for abnormal cell growth [37,38]. These results indicated that
developed nanoemulsion of CSB-INH could be administered orally for
the treatment of colon cancer.
N1 showed 96.90
6.02% cytotoxicity which was highly significant
than free CSB-INH (P b 0.05). The lowest molar concentration
(1.25 μM) of CSB-INH in N1 showed 8.42 1.60% cytotoxicity as com-
pared to only 0.14 0.03% cytotoxicity of free CSB-INH (Table 5). Over-
all, CSB-INH in the form of nanoemulsion was found to be highly potent
and efficacious than free CSB-INH on human colon cancer cell line.
These results indicated the suitability of developed nanoemulsion in
the treatment of colon cancer. The % viability of cells treated with free
CSB-INH was found to be 54.40% at a molar concentration of 100 μM
(Fig. 4). However, CSB-INH loaded nanoemulsion N1 showed significant
growth inhibiting activity on the same cells. The cell viability of N1
treated cells was found to be only 3.10% at a molar concentration of
100 μM which was highly significant than free CSB-INH (P b 0.05).
These results indicated the superiority of developed nanoemulsion as
compared to free CSB-INH. On the other hand, control formulation did
not inhibit cell growth at a greater extent (Fig. 4) which indicated that
developed nanoemulsion components were nontoxic. The concentra-
tions of free CSB-INH and N1 leading to 50% cell death (IC50) were also
determined by interpolation of concentration dependent cell viability
curves (Fig. 4). The IC50 value of free CSB-INH was found to be
100.72 μM. However, the IC50 value of CSB-INH in N1 was found to be
much lower than free CSB-INH (9.15 μM). Based on IC50 values, the
CSB-INH in developed nanoemulsion was found to be around 9 times
3.8. Pharmacokinetic study in rats
The in vivo pharmacokinetic studies were performed to quantify CSB-
INH in rat plasma after oral administration of CSB-INH nanoemulsion N1
and suspension formulation. The plasma concentration profiles of CSB-
INH in adult male albino Wistar rats following oral administration of
the optimized nanoemulsion (N1) and suspension formulation were
compared. The plasma concentration profile of CSB-INH for optimized
nanoemulsion represents a significantly greater improvement of CSB-
INH oral absorption than suspension formulation (Fig. 5). The results of
pharmacokinetic parameters of N1 and drug suspension are listed in
Table 6. The Tmax
was observed as 1.5 h for N1 and as well as for suspen-
sion formulation. However, the Cmax of N1 was found to be 452.19 ng/ml
as compared to 210.89 ng/ml of suspension formulation. Statistically, the
difference in Cmax of N1 was highly significant (P b 0.001) as compared to
suspension formulation. It was also observed that AUC0 − t and AUMC0 −
t of N1 were 1313.73 and 3870.57 ng·h/ml, respectively, which were
highly significant (P b 0.001) as compared to AUC0 − t (618.69 ng·h/
ml) and AUMC0
(2048.08 ng·h/ml) of suspension formulation.
−
t
Table 4
Correlation coefficients and release rate constants of CSB-INH nanoemulsions (N1—N5) and drug suspension.
Formulation
Zero order
K0
First order
k1
Higuchi
R2
Hixson–Crowell
Peppas
R2
R2
R2
R2
n
N1
N2
N3
N4
N5
Suspension
11.86
11.06
10.28
9.25
7.31
2.06
0.939
0.964
0.969
0.976
0.983
0.981
1.866
1.659
1.531
1.412
1.270
1.051
0.971
0.968
0.934
0.959
0.988
0.978
0.978
0.982
0.978
0.976
0.972
0.943
0.981
0.989
0.985
0.985
0.993
0.977
0.979
0.983
0.981
0.985
0.988
0.985
0.982
0.980
0.962
0.943
0.877
0.897
Correlation coefficient (R2), zero order rate constant (K0), first order rate constant (k1), and diffusion coefficient (n).

118
M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
Table 5
Table 6
Cytotoxicity (%) of free CSB-INH, optimized nanoemulsion (N1) and control on HT-29 hu-
man colon cancer cell lines (n = 3).
Pharmacokinetic parameters (mean SE, n = 6) of CSB-INH after an oral administration
of optimized nanoemulsion (N1) and CSB-INH suspension (5 mg/kg).
Concentration (μM)
Cytotoxicity (% SD)
Parameters
CSB-INH suspension
(Mean SE)
CSB-INH nanoemulsion
(Mean SE)
Free CSB-INH
N1
Control
Cmax (ng/ml)
Tmax (h)
210.89 (61.28)
1.5
452.19 (55.64)⁎⁎⁎
1.5
1313.73 (138.97)⁎⁎⁎
3870.57 (664.78)⁎⁎⁎
0.20 (0.01)
3.47 (0.18)
2.96 (0.32)
212.34
0
1.25
2.50
5.00
10.00
20.00
40.00
80.00
100.00
0
0
0
0.14 0.03
0.75 0.07
2.38 0.11
2.73 0.14
13.89 1.53
31.62 2.20
39.55 3.56
45.60 4.32
8.42 1.60
11.28 1.89
21.16 2.46
77.28 5.27
90.62 6.20
92.52 7.12
94.80 5.74
96.90 6.02
0.29 0.04
0.11 0.02
3.72 0.16
3.86 0.21
2.52 0.31
1.87 0.10
2.60 0.42
3.70 0.54
AUC0–24 (ng·h/ml)
AUMC0–24 (ng·h/ml)
618.69 (164.90)
2048.08 (512.74)
0.18 (0.01)
3.82 (0.27)
3.34 (0.24)
100
λz (h−1
(h)
)
T
½
MRT (h)
Relative bioavailability (%)
⁎p b 0.01 significant, ⁎⁎⁎p b 0.001) highly significant compared to CSB-INH suspension.
these results, it was concluded that the developed nanoemulsion might
be a promising vehicle for oral delivery of CSB-INH for the treatment of
colon cancer. For definitive preclinical results, further in vivo studies
will be performed in an appropriate animal colon cancer model to ascer-
tain efficacy in terms of reduction in tumor size.
However, the difference in the values of MRT was not statistically signif-
icant (P N 0.05) when the nanoemulsion and suspension formulations
were compared. The relative bioavailability of N1 with respect to suspen-
sion formulation was observed as 212.34%. The absorption of CSB-INH
from optimized nanoemulsion N1 resulted in a 2.12fold increase in bio-
availability as compared to suspension formulation. The enhanced bio-
availability of CSB-INH from nanoemulsion was probably due to lower
droplet size, higher drug release and the presence of surfactants such as
Tween-80 and Transcutol-HP in the formulations [26].
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgment
The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research and Research Center, College of Pharma-
cy, King Saud University.
4. Conclusions
In the present study, the carvone Schiff base of isoniazid was synthe-
sized and characterized for its possible anticancer potential. Various
nanoemulsion formulations of CSB-INH were developed, characterized,
and evaluated for in vitro cytotoxicity and in vivo pharmacokinetic stud-
ies. Based on the lowest droplet size (19.4 nm), least PI (0.189), lowest
viscosity (29.6 cP), optimal values of ZP (−30.8 mV) & RI (1.341), highest
% T (98.9%), highest drug release profile (94.5% after 24 h) and the pres-
ence of the lowest concentration of Triacetin (12% w/w), CSB-INH
nanoemulsion N1 was selected for in vitro cytotoxicity and in vivo phar-
macokinetic studies. In vitro cytotoxicity studies on HT-29 human colon
cancer cells indicated that CSB-INH in optimized nanoemulsion was
around seven times more efficacious than free CSB-INH in terms of cell
growth inhibiting activity. Moreover, pharmacokinetic studies in rats
showed rapid absorption of CSB-INH from optimized nanoemulsion as
compared to its suspension formulation. The oral bioavailability of CSB-
INH was significantly higher than its suspension formulation. From
References
[1] H.S.N. Kumar, T. Parumasivam, F. Jumaat, P. Ibrahim, M.Z. Asmawi, A. Sadikun, Syn-
thesis and evaluation of isonicotinoyl hydrazone derivatives as antimycobacterial
and anticancer agents, Med. Chem. Res. 23 (2014) 269–279.
[2] O.H. Sjo, M. Berg, M. Merok, M. Kolberg, A. Svindland, R.A. Lothe, A. Nesbakken, Peri-
toneal carcinomatosis of colon cancer origin: highest incidence in women and in pa-
tients with right-sided tumors, J. Surg. Oncol. 104 (2011) 792–797.
[3] P. Yin, Y. Wang, Y. Qiu, L. Hou, X. Liu, J. Qin, Y. Duan, P. Liu, M. Qiu, Q. Li, Bufalin-
loaded mPEG-PLGA-PLL-cRGD nanoparticles: preparation, cellular uptake, distribu-
tion and anticancer activity, Int. J. Nanomedicine 7 (2012) 3961–3969.
[4] D.M. Parkin, F. Bray, J. Ferlay, P. Pisani, Global cancer statistics, 2002, CA Cancer J.
Clin. 55 (2005) 74–108.
[5] Z. Rahman, K. Kohli, R.K. Khar, M. Ali, N.A. Charoo, A.A. Shamsher, Characterization
of 5-fluorouracil microspheres for colonic delivery, AAPS PharmSciTech 7 (2006)
E47.
[6] M.A. Bhat, M.A. Al-Omar, Synthesis, characterization and in vitro anti-Mycobacteri-
um tuberculosis activity of terpene Schiff bases, Med. Chem. Res. 22 (2013)
4522–4528.
[7] N. Demirbas, S.A. Karaoglu, A. Demirbas, K. Sancak, Synthesis and antimicrobial ac-
tivities of some new 1-(5-phenylamino-[1,3,4] thiadiazol-2-yl) methyl-5-oxo-
[1,2,4] triazole and 1-(4-phenyl-5-thioxo-[1,2,4] triazol-3-yl) methyl-5-oxo-[1,2,4]
triazolederivatives, Eur. J. Med. Chem. 39 (2004) 793–804.
[8] P. Vicini, M. Incerti, I.A. Doytchinova, P. La Colla, B. Busonera, R. Loddo, Synthesis and
anti proliferative activity of benzo[d]isothiazole hydrazones, Eur. J. Med. Chem. 41
(2006) 624–632.
[9] K. Sztanke, T. Tuzimski, J. Rzymowska, K. Pasternak, A.M. Kandefer-Szersze, Synthe-
sis, determination of the lipophilicity, anticancer and antimicrobial properties of
some fused 1,2,4-triazole derivatives, Eur. J. Med. Chem. 43 (2008) 404–419.
[10] T.J. de Faria, M. Roman, N.M. de Souza, R.D. Vecchi, J.V. de Assis, A.L.G. dos Santos, I.H.
Bechtold, N. Winter, M.J. Soares, L.P. Silva, M.V. De Almeida, A. Bafica, An isoniazid
analogue promotes Mycobacterium tuberculosis-nanoparticle interactions and en-
hances bacterial killing by macrophages, Antimicrob. Agents Chemother. 56
(2012) 2259–2267.
[11] F.A.R. Rodrigues, A.C.E. Oleveira, B.C. Cavalcanti, C. Pessoa, A.C. Pinheiro, M.N.V. De
Souza, Biological evaluation of isoniazid derivatives as an anticancer class, Sci.
Pharm. 82 (2014) 21–28.
[12] H. Kumar, D. Malhotra, R. Sharma, E. Sausville, M. Malhotra, Synthesis, characteriza-
tion and evaluation of isoniazid analogues as potent anticancer agents,
Pharmacologyonline 3 (2011) 337–343.
[13] F.K. Alanazi, N. Haq, A.A. Radwan, I.A. Alsarra, F. Shakeel, Formulation and evaluation
of cholesterol-rich nanoemulsion (LDE) for drug delivery potential of cholesteryl-
maleoyl-5-fluorouracil, Pharm. Dev. Technol. 20 (2015) 266–270.
[14] F.K. Alanazi, N. Haq, A.A. Radwan, I.A. Alsarra, F. Shakeel, Cholesterol-rich
nanoemulsions (LDE) for drug targeting of cholesteryl-succinyl-5-fluorouracil con-
jugate, Curr. Nanosci. 10 (2014) 287–291.
Fig. 5. Plasma concentration–time profile curves of CSB-INH after oral administration of
optimized nanoemulsion and suspension formulations in adult male Albino rats
(Mean SD, n = 6 and dose 5 mg/kg).

M.A. Bhat et al. / Journal of Molecular Liquids 203 (2015) 111–119
119
[15] F. Shakeel, N. Haq, A. Al-Dhfyan, F.K. Alanazi, I.A. Alsarra, Chemoprevention of skin
cancer using low HLB surfactant nanoemulsion of 5-fluorouracil: a preliminary
study, Drug Deliv. (2013). http://dx.doi.org/10.3109/10717544.2013.868557.
[16] J.A. Moura, C.J. Valduga, E.R. Tavares, I.F. Kretzer, D.A. Maria, R.C. Maranhão, Novel
formulation of a methotrexate derivative with a lipid nanoemulsion, Int. J.
Nanomedicine 6 (2011) 2285–2295.
[27] F. Shakeel, S. Shafiq, N. Haq, F.K. Alanazi, I.A. Alsarra, Nanoemulsions as potential ve-
hicles for transdermal and dermal delivery of hydrophobic compounds: an over-
view, Expert Opin. Drug Deliv. 9 (2012) 953–974.
[28] F. Shakeel, N. Haq, M. Elbadry, F.K. Alanazi, I.A. Alsarra, Ultra fine super
selfnanoemulsifying drug delivery system (SNEDDS) enhanced solubility and disso-
lution of indomethacin, J. Mol. Liq. 180 (2013) 89–94.
[17] C.H. Azevedo, J.P. Carvalho, C.J. Valduga, R.C. Maranhão, Plasma kinetics and uptake
by the tumor of a cholesterol-rich microemulsion (LDE) associated to etoposide ole-
ate in patients with ovarian carcinoma, Gynecol. Oncol. 97 (2005) 178–182.
[18] D.G. Rodrigues, D.A. Maria, D.C. Fernandes, C.J. Valduga, R.D. Couto, O.C.M. Ibanez,
R.C. Maranhao, Improvement of paclitaxel therapeutic index by derivatization and
association to a cholesterol-rich microemulsion: in vitro and in vivo studies, Cancer
Chemother. Pharmacol. 55 (2005) 565–576.
[29] F. Shakeel, N. Haq, F.K. Alanazi, I.A. Alsarra, Impact of various nonionic surfactants on
self-nanoemulsification efficiency of two grades of Capryol (Capryol-90 and
Capryol-PGMC), J. Mol. Liq. 182 (2013) 57–63.
[30] H. Yu, Q. Huang, Improving the oral bioavailability of curcumin using novel
organogel-based nanoemulsions, J. Agric. Food Chem. 60 (2012) 5373–5379.
[31] P. Costa, J.M.S. Lobo, Modeling and comparison of dissolution profiles, Eur. J. Pharm.
Sci. 15 (2001) 123–133.
[19] M.L. Dias, J.P. Carvalho, D.G. Rodrigues, S.R. Graziani, R.C. Maranhão, Pharmacokinet-
ics and tumor uptake of a derivatized form of paclitaxel associated to a cholesterol-
rich nanoemulsion (LDE) in patients with gynecologic cancers, Cancer Chemother.
Pharmacol. 59 (2007) 105–111.
[20] R. Pandey, A. Zahoor, S. Sharma, G.K. Khuller, Nanoparticle encapsulated antituber-
cular drugs as a potential oral drug delivery system against murine tuberculosis, Tu-
berculosis 83 (2003) 373–378.
[21] A. Sharma, R. Pandey, S. Sharma, G.K. Khuller, Chemotherapeutic efficacy of poly(dl-
lactide-co-glycolide) nanoparticle encapsulated antitubercular drugs at subthera-
peutic dose against experimental tuberculosis, Int. J. Antimicrob. Agents 24 (2004)
599–604.
[22] P. Blasi, A. Schoubben, S. Giovagnoli, C. Rossi, M. Ricci, Fighting tuberculosis: old
drugs, new formulations, Expert Opin. Drug Deliv. 6 (2009) 977–993.
[23] A. Sosnik, A.M. Carcaboso, R.J. Glisoni, New old challenges in tuberculosis: potential-
ly effective nanotechnologies in drug delivery, Adv. Drug Deliv. Rev. 62 (2010)
547–559.
[32] S. Dash, P.N. Murthy, L. Nath, P. Chowdhury, Kinetic modeling on drug release from
controlled drug delivery systems, Acta Pol. Pharm. 67 (2010) 217–223.
[33] M. Iqbal, M.A. Bhat, F. Shakeel, Development and validation of UHPLC-MS/MS assay
for rapid determination of a carvone Schiff base of isoniazid (CSB-INH) in rat plas-
ma: application to pharmacokinetic study, Biomed. Chromatogr. (2014). http://
dx.doi.org/10.1002/bmc.3368.
[34] M.K. Anwer, S. Jamil, E.O. Ibnouf, F. Shakeel, Enhanced antibacterial effects of clove
essential oil by nanoemulsion, J. Oleo Sci. 63 (2014) 347–354.
[35] O. Nuchuchua, U. Sakulku, N. Uawongyart, S. Puttipipatkhachorm, A. Soottitantawat,
N. Ruktanonchai, In vitro characterization and mosquito (Aedes aegypti) repellent
activity of essential-oils-loaded nanoemulsions, AAPS PharmSciTech 10 (2009)
1234–1242.
[36] M. El-Badry, N. Haq, G. Fetih, F. Shakeel, Solubility and dissolution enhancement of
tadalafil using self-nanoemulsifying drug delivery system, J. Oleo Sci. 63 (2014)
567–576.
[37] A. Kajal, S. Bala, S. Kamboj, N. Sharma, V. Saini, Schiff base:
pharmacophore, J. Catal. 2013 (2013) (E893512).
a versatile
[24] R. Pandey, Z. Ahmad, Nanomedicine and experimental tuberculosis: facts, flaws, and
future, Nanomedicine 7 (2011) 259–272.
[25] A. Dorhoi, S.T. Reece, S.H. Kaufmann, For better or for worse: the immune response
against Mycobacterium tuberculosis balances pathology and protection, Immunol.
Rev. 240 (2011) 235–251.
[38] A.J. Guo, X.S. Xu, Y.H. Hu, M.Z. Wang, X. Tan, Effects of ternary complexes of copper
with salicylaldihyde-amino acid Schiff base coordination compounds on the prolif-
eration of BGC823 cells, Chin. J. Cancer 29 (2010) 277–282.
[26] S. Shafiq, F. Shakeel, S. Talegaonkar, F.J. Ahmad, R.K. Khar, M. Ali, Development and
bioavailability assessment of ramipril nanoemulsion formulation, Eur. J. Pharm.
Biopharm. 66 (2007) 227–243.