pharmaceutics

Article
Formulation and Characterization of Eplerenone
Nanoemulsion Liquisolids, An Oral Delivery System
with Higher Release Rate and
Improved Bioavailability
Ahmed Khames 1,2
1 Department of pharmaceutics and industrial pharmacy, Beni-suef University, Beni-Suef 62514, Egypt;
[email protected]
2 Department of pharmaceutics and industrial pharmacy, Taif University, Taif 21944, Saudi Arabia




Received: 3 December 2018; Accepted: 16 January 2019; Published: 18 January 2019


Abstract: Because Eplerenone (EPL) is a Biopharmaceutical Classification System (BCS) class-II

drug and is prone to extensive liver degradation, it suffers from poor bioavailability after oral
administration. This work aimed to prepare liquisolids loaded with EPL-nanoemulsions (EPL-NEs)
that have a higher drug release rate and improved bioavailability by the oral route. Based on
solubility studies, mixtures of Triacetin (oil) and Kolliphor EL/PEG 400 surfactant/co-surfactant
(Smix ) in different ratios were used to prepare EPL-NE systems, which were characterized and
optimized for droplet size, zeta potential, polydispersity index (PDI), and drug content. Systems were
then loaded onto liquisolid formulations and fully evaluated. A liquisolid formulation with better
drug release and tableting properties was selected and compared to EPL-NEs and conventional EPL
oral tablets in solid-state characterization studies and bioavailability studies in rabbits. Only five NEs
prepared at 1:3, 1:2, and 3:1 Smix met the specified optimization criteria. The drug release rate from
liquisolids was significantly increased (90% within 45 minutes). EPL-NE also showed significantly
improved drug release but with a sustained pattern for four hours. Liquisolid bioavailability reached
2.1 and 1.2 relative to conventional tablets and EPL-NE. This suggests that the EPL-NE liquisolid is
a promising oral delivery system with a higher drug release rate, enhanced absorption, decreased
liver degradation, and improved bioavailability.

Keywords: eplerenone; nanoemulsion; liquisolids; Triacetin; bioavailability

1. Introduction
Eplerenone (EPL), a member of the spironolactone group, is a new selective steroidal
anti-mineralocorticoid receptor blocker. EPL selectively prevents aldosterone receptor binding
in both epithelial and non-epithelial tissues that results in the blockage of the renin-angiotensin-
aldosterone-system (RAAS), with subsequent inhibition of sodium reabsorption that affects blood
pressure and the cardiovascular system [1–3]. The resultant extended increase of plasma renin and
serum aldosterone also inhibits the negative regulatory feedback of aldosterone on renin release [4,5].
EPL is used to treat hypertension, central serous retinopathy, and chronic heart failure either alone
or in combination with other antihypertensive agents [6–8]. EPL is an aldosterone analogous with
efficient renal and cardiac protective abilities, which is why EPL is used by elderly patients taking
Angiotensin-Converting Enzyme Inhibitors (ACEIs) or Angiotensin II Receptor Blockers (ARBs) [9].
EPL also helps to reduce the acute myocardial infarction death rate in patients with systolic dysfunction
of the left ventricle and heart failure [10,11].

Pharmaceutics 2019, 11, 40; doi:10.3390/pharmaceutics11010040 www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2019, 11, 40 2 of 18

EPL (molecular weight of 414.49 g/mol) is a crystalline powder that is slightly soluble in water
(<1 mg/mL). It has a high octanol/water partition coefficient (log Kow = 7.1 at pH 7.0). EPL is a BCS
Class II drug (low solubility and high permeability) of low bioavailability that is significantly affected
by drug solubility [12].
After oral administration and once dissolved, EPL is rapidly absorbed from the upper
gastrointestinal tract (GIT) with a negligible food effect and reaches its maximum plasma concentration
within 1.5–2 hours. EPL is extensively metabolized in the liver (less than 5% of a dose is excreted
in urine in an unmodified form) via CYP3A4 to inactive metabolites [1,13]. The hepatic first-pass
metabolism of EPL is 12.6% and 27.1% under fasted and fed conditions, respectively, with a significant
effect on drug bioavailability (69% for a 100 mg oral tablet). EPL is mainly excreted in urine with a t1/2
of 3 to 6 hours [14].
For efficient absorption from the GIT, EPL must reach the absorption site in a soluble form; thus,
poor solubility is the main factor that retards drug absorption, decreases bioavailability, and may
increase the probability of drug damage in the GIT [15]. Improving drug solubility is a major concern
of drug formulators, especially for drugs classified as BCS class II drugs in which the dissolution rate
represents the rate determining step of the absorption process [16,17]. Several techniques have been
studied and used to increase the solubility of lipophilic drugs [16,18].
Lipid-based drug delivery systems, especially nano-sized systems including nano-lipid
carriers (NLCs), solid lipid nanoparticles (SLNs), self-emulsifying drug delivery systems (SEDDSs),
and nanoemulsions (NEs). Since nano-sized systems can easily mix with GIT fluids to increase
drug solubility that results in enhanced absorption and improved bioavailability, they have received
extensive consideration for oral delivery of poorly soluble drugs [19]. Many studies have claimed that
lipid formulations escape the dissolution step that is necessary for conventional solid or suspension
oral dosage forms and pass through GIT membranes via the lymphatic system, which keeps the drug
away from enzymatic degradation that occurs in the hepatic first-pass effect [20,21]. This is a unique
advantage for BCS class II drugs, especially those that are susceptible to enzymatic liver degradation,
which significantly improves their absorption and therefore their bioavailability.
NEs are lipid-based colloidal systems that are transparent and thermodynamically stable,
with a droplet size in the range of 50–200 nm. NEs are stabilized by a surfactant/co-surfactant
interfacial film that results in ultra-low interfacial tensions and high kinetic stability that prevents
droplet coalescence and phase separation. In addition to high physical stability, other advantages
of NEs include high solubilization ability, efficient membrane permeability, superior protection of
drugs against oxidation, hydrolytic and physiological enzymatic degradation, controlled release, drug
targeting, and increased bioavailability. Ease of preparation with a high cost benefit increases the
potential use of NEs in pharmaceutical applications [22,23].
The liquisolid mixture has a dry appearance and is a non-adherent and flowable powder mixture
containing liquid medication that is suitable for direct compression [24]. A liquisolid mixture is
prepared by simple blending of a liquid medication with selected powder excipients referred to as the
carrier and coating materials. Various grades of cellulose, starch, and lactose may be used as the carrier,
while very fine silica powder is usually used as a coating material. The term “liquid medication” refers
to drug solutions, drug suspensions, emulsions, or liquid oily drugs. Quantities of carrier and coating
materials needed to produce adequately flowing and compressible properties are calculated according
to a liquisolid formation mathematical model [25]. According to this model, the carrier and coating
materials can retain only certain amounts of liquid (maximum liquid load) while keeping acceptable
flow and compression properties, which is called the liquid load factor (Lf ). Depending on the selected
excipient ratio (R) and the calculated liquid load factors, the required amounts of carrier and coating
materials can be calculated [26].
A previous study [15] indicated that solubility enhancement of BCS class II drugs has a positive
effect of on bioavailability at the main absorption site. The present study aimed to prepare EPL-NE
loaded liquisolid tablet mixtures that combined the advantages of the NE system (i.e., higher drug
Pharmaceutics 2019, 11, 40 3 of 18

absorption due to the surfactant effect on GIT permeability [27] and protection from liver degradation
via para-cellular penetration [28]) with the advantages of liquisolids (i.e., rapid, high drug release at
the main absorption site and presentation of NE formulations in a free flowing, highly compressible
dry form suitable for oral delivery with enhanced drug bioavailability). Based on drug solubility
studies, Triacetin, Kolliphor EL, and PEG 400 as an oil, surfactant, and co-surfactant, respectively
were selected for preparing NE systems by the titration method. Pseudo-ternary phase diagrams
were constructed to determine the area of clarity (NE area) and different plain NE systems were
prepared using different Smix ratios. Based on droplet size, zeta potential, and polydispersity index
(PDI), NE systems were optimized for drug loading and were incorporated into liquisolid formulation
prepared using Avicel/nano-silica as a carrier/coat mixture according to the proposed mathematical
model. EPL-NE liquisolids were characterized before and after compression and drug release was
compared to that of an unformulated EPL. An EPL-NE liquisolid formulation with higher drug release
and better tableting properties was selected and subjected to further solid state characterization and
bioavailability studies in comparison to EPL-NEs and conventional EPL oral tablets in rabbits.

2. Materials and Methods

2.1. Materials
EPL and valdecoxib (Sigma-Aldrich, Steinheim, Germany), Avicel PH 102 (FMC Corp.,
Philadelphia, PA, USA), nanometer-sized amorphous silicon dioxide (SiO2), Triacetin, olive oil, oleic
acid, sesame oil, soybean oil, isopropyl myristate (IPM), and isopropyl propionate (IPP) (Sigma-Aldrich,
Steinheim, Germany), Transcutol HP, Labrasol, and Labrafil (gift from Gattefosse SAS, Saint-Priest
Cedex, France), Pharmaburst 500 (SPI Pharma, Inc. Wilmington, DE, USA), Poly ethylene glycol 400
(PEG 400), tween 40, tween 80, Kolliphor EL, and propylene glycol (PG) (Merck KGaA, Darmstadt,
Germany), all other solvents are HPLC grade (Sigma-Aldrich, Steinheim, Germany).

2.2. Methodology

2.2.1. Screening of EPL Solubility in NE Components

The solubility of EPL in different lipids (olive oil, oleic acid, sesame oil, soybean oil, IPM,
IPP, Labrafil, and Triacetin), surfactants (Labrasol, Tween 40, Tween 80, and Kolliphor EL),
and co-surfactants (PEG 400, ethanol, PG, Transcutol HP, and glycerol) was investigated by separately
adding excess amounts of EPL to 1 mL of the tested vehicle in small eppendorf. Mixtures were
shaken for 72 hours (Hicon, New Delhi, India) at 25 ± 1.0 ◦ C to reach the equilibrium. Samples
were centrifuged (REMI Pvt. Ltd., Mumbai, India) at 14,000 rpm for 15 minutes, the supernatant was
collected and filtered (0.2 µm membrane), and drug concentration was determined (in triplicate) by
a validated HPLC method. Drug solubility was recorded as mg/ml concentrations [2].

2.2.2. Construction of Pseudo-Ternary Phase Diagrams

For construction of the pseudo-ternary phase, the titration method (spontaneous emulsification)
was applied as follows: The selected surfactant and co-surfactant were combined in different weight
ratios (Smix ) (coded as: S1 (3:1), S2 (2:1), S3 (1:1), S4 (1:2), and S5 (1:3)). The selected oil was added to
each Smix in different ratios (oil:Smix ratios of 0:100, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30,
80:20, 90:10, and 100:0) in screw-capped vials and mixed at room temperature until a clear solution was
obtained. Phase diagrams were constructed by titrating these samples with aliquots of double-distilled
water with continuous stirring for a sufficient amount of time to reach equilibrium. Samples were
visually examined for clarity and subsequently classified as being either a clear NE, an emulsion, or
gel (if present). The phase behavior of the systems was mapped on triangle phase diagrams with three
apices representing Smix , oil, and water. The area of clear NE was determined.
Pharmaceutics 2019, 11, 40 4 of 18

2.2.3. Optimization of NE Formulations

After the o/w NE regions in the phase diagrams were identified, 20 plain NE formulations were
prepared using different oil:Smix :water ratios within the NE region. Plain systems that had a droplet
size in the range of 25 to 50 nm, PDI less than 0.3, zeta potential in the range of 25–35 mV, and retained
transparency for one week at room temperature were optimized and selected for loading the drug.

2.2.4. Loading of EPL onto NE Formulations

For EPL loading onto NE formulations, an excess amount of drug (150 mg) was added to the
optimized oil/Smix mixture and constantly stirred using a mechanical stirrer until a homogenous
mixture was obtained. The required amount of water was added in a drop-wise manner with gentle
agitation until a clear loaded EPL-NE was produced.

2.2.5. Characterization of EPL-NE Formulations

Drug Loading
To determine the drug loading capacity, EPL-NE formulations were stirred for 24 hours at
25 ± 1.0 ◦ C. Un-dissolved drug was removed by centrifugation at 20,000 rpm for 15 minutes,
the supernatant was isolated and filtered (0.2 µm membrane), and the drug content was determined
using an HPLC assay method [2]. The analyses were performed in triplicate.

Droplet Size, Size Distribution, and Z-Potential Measurement

The average droplet diameter, polydispersity index (PDI), and the droplet surface charge (zeta
potential) of the EPL-NE formulations were investigated using photon correlation spectroscopy
(Zetasizer 4, Malvern, UK) at 90 ◦ C and 25 ◦ C after dilution with ultra-purified water to an adequate
intensity. Each measurement was performed at least in triplicate. The pH value of the samples was
maintained at 6.5 ± 0.5.

2.2.6. Loading of Optimized EPL-NE Systems onto Liquisolid Mixtures

Calculation of Carrier and Coating Material Amounts

EPL-NE liquisolids were prepared using Avicel PH 102/nano-sized amorphous silicon dioxide
as carrier/coat mixtures with a fixed ratio (R) equals 20 that was recommended to give a satisfactory
hardness and enhanced release profiles [29]. According to the mathematical model described by
Spireas and Bolton [30], the Φ-values of the carrier and the coating materials were calculated for each
dry EPL-NE formulation and the calculated Lf -values (the weight ratio of the liquid medication and
carrier powder) were used to determine the amount of carrier, while the amount of coating material
was calculated from the applied R-value. These calculated amounts of the carrier and coating materials
were predicted to produce formulation mixtures with good flow and compression properties.

Preparation of EPL-NE Liquisolids

In a porcelain mortar, dry EPL-NE formulation was manually mixed with carrier material for
a suitable amount of time to ensure efficient liquid absorption. The coating material was then added
and mixing continued to adsorb excess fluid. This mixing order favors optimal drug release based
on a previous publication [31]. Finally, the prepared liquisolid mixtures were directly compressed on
a suitable punch-die set (single punch eccentric tablet press EP-1, Erweka, Heusenstamm, Germany)
using 10% Pharmaburst 500 SPI (as disintegrant) and 1% magnesium stearate (as a lubricant).
Formulations composition is presented in Section 3.4 A batch of 10 EPL-NE loaded liquisolid
formulation tablets was prepared.
Pharmaceutics 2019, 11, 40 5 of 18

2.2.7. Evaluation of EP-NE Liquisolids

Pre-Compression Evaluation
Flow and compression properties of the prepared EPL-NE loaded liquisolid mixtures were
evaluated by measuring the angle of repose (θ), Carr’s index (Ci ), and Hausner ratio [24]. The reported
value is an average of three measurements.

Post-Compression Evaluation
The prepared EPL-NE loaded liquisolids were evaluated for weight variation, content
uniformity [2], hardness (TBH 325, Erweka, Heusenstamm, Germany), friability (TAR, Erweka,
Heusenstamm, Germany), and disintegration time (DST-3/6 automated disintegration tester, Logan,
UT, USA) according to standardized United States Pharmacopeia (USP) conditions [32].

2.2.8. In-Vitro Drug Release Studies

Drug release properties of the prepared EPL-NE loaded liquisolids were studied using the USP XXIV
dissolution testing apparatus II (UDT-804 paddle dissolution apparatus, Logan, UT, USA) in 500 mL of
0.1 N HCl (pH 1.2) as the dissolution medium at 37 ± 0.5 ◦ C and 50 rpm. At specified time intervals,
a 5 mL sample was withdrawn with replacement and the cumulative percentage of drug release was
calculated using an HPLC method [2]. For comparison, the drug release rate from corresponding EPL-NE
formulations (using dialysis bags with a 12 kDa molecular weight cutoff) and the dissolution rate of
unformulated EPL was also determined. The mean of six determinations was recorded.

Kinetic Modeling of Release Data

For quantitative kinetic analysis of EPL release profiles from optimized NEs and NE liquisolids,
KinetDS 3.0 (Aleksander Mendyk, GNU GPLv3 license, 2007, Kraków, Poland) software was used to
investigate the best data fit according to zero order, first order, Higuchi, Korsmeyer-Peppas, and Weibull
diffusion models.
Based on previous in-vitro characterization studies, an EPL-NE liquisolid tablet formulation
that showed good flow and compression properties and a high drug release rate were selected and
subjected to further solid-state characterization and bioavailability studies.

2.2.9. Solid State Characterizations and Compatibility Studies

Samples of EPL-NE liquisolid formulation, unformulated EPL, and physical mixture were
subjected to the following studies described below.

Differential Scanning Calorimetry (DSC)

Samples (2–4 mg) were subjected to thermal analysis using a differential scanning calorimeter
(Perkin-Elmer DSC4, Shelton, CT, USA.) over a temperature range of (30–300 ◦ C) and a heating rate of
10 ◦ C/minute. DSC thermograms were recorded and analyzed.

Infrared Spectroscopy (IR)

Samples were scanned in the range of 4000–500 cm−1 at ambient temperature using an IR
spectrophotometer (Shimadzu IR-435, Kyoto, Japan), after compression into a transparent disc with
KBr 10,000 to 15,000 pounds/inch2 ). IR spectra were recorded and analyzed.

2.2.10. In-Vivo Pharmacokinetic Studies

The selected EPL-NE loaded liquisolid formulation (treatment A) was compared to the
corresponding EPL-NE formulation (treatment B) and conventional oral tablets prepared using the
unformulated drug and the same liquisolid formulation excipients (treatment C), in rabbits.
Pharmaceutics 2019, 11, 40 6 of 18

Study Design
A single dose bioavailability study with a group of six healthy male albino rabbits (2.5–3.0 kg)
was conducted in two phases based on a randomized crossover design. The rabbits were kept on
the same diet during the study, with overnight fasting prior to drug administration but free access to
water. Rabbits were cannulated through the marginal air vein to collect blood samples. Control plasma
samples (2 mL) were withdrawn from each rabbit before drug administration.
A suitable weight of each treatment equivalent to the calculated animal dose based on the Paget
and Barnes table [33] was suspended in a minimal volume of water and orally administered to each
animal with the aid of gastric gavage.
Blood samples were collected at pre-determined time intervals for 24 hours following dosing.
Blood was collected in heparinized tubes and immediately centrifuged at 3500 rpm for 15 minutes.
Plasma was transferred to labeled capped tubes and stored at −20 ◦ C until analysis.

Determination of EPL in Plasma

The drug concentration in the collected plasma samples was determined using a sensitive,
validated RP-HPLC assay [34], briefly described as follows:

Chromatographic Conditions
The mobile phase, consisting of acetonitrile: Water (50:50, v/v), was injected at a flow rate of
1 mL/minute at room temperature in the presence of valdecoxib as an internal standard. A UV detector
at λmax 241 nm was used to quantify EPL.

Sample Preparation for Determination of EPL

Frozen plasma samples were thawed at room temperature and were prepared as follows: In 15 mL
stoppered test tubes, each plasma sample (1 mL) was added to 50 µL of internal standard solution
(10 µg/mL), followed by vortex mixing for one minute. Dichloromethane:diethyl ether [(4:6, v/v),
5 mL] was added and the mixture was shaken for 30 minutes followed by centrifugation at 3000 rpm
for 10 minutes. The organic layer was transferred into a new tube and was evaporated to dryness under
a nitrogen stream. Finally, the residue was reconstituted in mobile phase (250 µL) and was injected
into a chromatographic apparatus. EPL concentration was calculated based on the data obtained from
the constructed calibration curve in plasma.

Pharmacokinetic Calculations
Plasma concentration time curves were constructed and the data were manipulated using
WinNonlin Professional 4.0.1 software (Pharsight Corp., Cary, NC, USA), followed by calculation
of pharmacokinetic parameters (Cmax (ng/mL), Tmax (hour), AUC0–24 and AUC0–∞ (ng·hour/mL,
Kel (hours−1 ), and t1/2 (hours)) for each rabbit.

Statistical Analysis of Pharmacokinetic Data

The mean pharmacokinetic parameters were statistically analyzed using the post hoc one-way
ANOVA test (Tukey mode) at a p-value > 0.05 and confidence intervals were calculated (IBM-SPSS,
Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Screening of EPL Solubility in NE Components

Selection of appropriate NE formulation components is a significant parameter to obtain a stable
system [35]. For hydrophobic drugs, lipid solubility is more important than other factors since it
directly affects drug loading and hence the formulation volume for the required dosage. In addition,
Pharmaceutics 2019, 11, 40 7 of 18

if drug loading is dependent on adding surfactant and/or co-surfactant, the drug will be prone
to precipitation when diluted with GIT due to inhibition of the surfactant/co-surfactant solvation
effect [35,36]. Moreover, for oil with low drug solubilizing ability, larger oil amounts may be needed
Pharmaceutics 2018, 10, x FOR PEER REVIEW 7 of 18
to tolerate the required drug dose. To sequentially attain stable systems, a higher concentration of
surfactant is used,
tolerate which may
the required drugaffect
dose.the
Tosafety of the NE
sequentially system
attain stable[37]. In this
systems, work, concentration
a higher an oil with maximum
of
surfactantwas
drug solubility is used, whichtomay
selected affectNE
prepare thesystems.
safety of The
the NE system of
solubility [37].
EPLIn in
thisTriacetin
work, an(10.59
oil with
mg/mL)
maximum
was higher thandrug solubility
in the was selected
other tested to prepare
oils (Figure 1), NE systems.
which The solubility
correlated with theof EPL in Triacetin
medium chain fatty
(10.59 mg/ml) was higher than in the other tested oils (Figure 1), which correlated with the medium
acids and small molar volume of the Triacetin triglyceride that support EPL solubilization [38]. Thus,
chain fatty acids and small molar volume of the Triacetin triglyceride that support EPL solubilization
Triacetin was selected as the oily phase for preparation of NE systems.
[38]. Thus, Triacetin was selected as the oily phase for preparation of NE systems.

12

10.59

10
EPL Solubility (mg/ml)

8
6.8

6
4.8
4.12
4 3.6
3.12
2.11 1.97 2.1
2 1.4
1.2
0.7 0.8
0.25 0.39 0.26 0.3
0

Tested liquid

Figure
Figure 1. 1. Solubilityof
Solubility of EPL
EPL in
indifferent
differentvehicles.
vehicles.

The selection of an appropriate SAA for NE formulation depends mainly on its safety,
The selection of an appropriate SAA for NE formulation depends mainly on its safety,
compatibility, and ability to lower the surface free energy at the water/oil interface to produce
compatibility, and ability to lower the surface free energy at the water/oil interface to produce
physically stable systems [36]. Non-ionic SAAs show relatively low physiological toxicity as
physically stable
compared systems
to their [36]. Non-ionic
ionic equivalents. Non-ionic SAAs
SAAs show relatively
also exhibit low physiological
emulsification toxicity as
properties at a lower
compared to their ionic
concentration, equivalents.
as indicated Non-ionic
by a relatively lowerSAAs also
critical exhibit
micelle emulsification
concentration, whichproperties
is favorableatfora lower
concentration, as indicated
oral administration, by a relatively
as surfactants in highlower critical micelle
concentrations result inconcentration,
GIT irritation. Inwhich
addition,is favorable
for o/w for
NE systems, a hydrophilic
oral administration, as surfactants nonionic
in highSAA with a higher result
concentrations HLB value
in GIT (>10) shows a In
irritation. higher positive
addition, for o/w
surface a
NE systems, excess that results
hydrophilic in accumulation
nonionic SAA with at the oil/water
a higher interface,
HLB valuewith (>10)an shows
efficientadecrease
higher in positive
surface tension and improved system stability [35]. In this work, four nonionic SAAs with a high HLB
surface excess that results in accumulation at the oil/water interface, with an efficient decrease in
were evaluated for their suitability in the formulation of EPL-NE systems, based on their solubility.
surfaceKolliphor
tension and improved
EL displayed system EPL
maximum stability [35].(6.8
solubility In mg/ml)
this work,
withfour
a high nonionic
HLB (HLB SAAs= 14)with a high
(Figure 1), HLB
were evaluated for their suitability in the formulation of EPL-NE
which made it the most suitable SAA for preparing stable EPL-NE systems. systems, based on their solubility.
Kolliphor EL displayed
In order to reducemaximum
oil/waterEPL solubility
interfacial (6.8
tension mg/mL) with
significantly a high interfacial
and increase HLB (HLB = fluidity,
film 14) (Figure 1),
a combination of high HLB surfactants and low HLB
which made it the most suitable SAA for preparing stable EPL-NE systems. co-surfactants is preferable to use of a single
In order to reduce oil/water interfacial tension significantly and increase interfacialthat
SAA. Co-surfactants are usually short or medium chain alcohols (C3–C8) that are added thus filmthe
fluidity,
amount of SAA used can be decreased. Co-surfactants are also distributed between SAA molecules
a combination of high HLB surfactants and low HLB co-surfactants is preferable to use of a single
at the oil/water interface that lowers the interaction between polar heads with a subsequent increase
SAA. Co-surfactants are usually short or medium chain alcohols (C3–C8) that are added thus that the
in interfacial film flexibility, which efficiently protects the oil droplet and increases hydrocarbon tail
amountoilofpenetration
SAA usedtocan form bea decreased. Co-surfactants
stable film [39]. Based on drugare also distributed
solubility results, PEGbetween SAA molecules
400 was selected to be at
the oil/water interface
used as the that lowers
co-surfactant for EPL-NEthe formulations.
interaction between polar heads with a subsequent increase
in interfacial filmonflexibility,
Based the resultswhich
of EPL efficiently protectsin
solubility presented the oil droplet
Figure and Kolliphor
1, Triacetin, increasesEL, hydrocarbon
and PEG tail
400 showed
oil penetration maximum
to form drugfilm
a stable solubility and were
[39]. Based selected
on drug as the oil,
solubility surfactant,
results, PEGand 400 co-surfactant,
was selected to be
used asrespectively, for the preparation
the co-surfactant for EPL-NE of NE formulations.
formulations.
Pharmaceutics 2019, 11, 40 8 of 18

Based on the results of EPL solubility presented in Figure 1, Triacetin, Kolliphor EL, and PEG
400 showed maximum drug solubility and were selected as the oil, surfactant, and co-surfactant,
respectively, for the preparation of NE formulations.
Pharmaceutics 2018, 10, x FOR PEER REVIEW 8 of 18
3.2. Pseudo-Ternary Phase Diagram Study
3.2. Pseudo-Ternary Phase Diagram Study
Using Triacetin as oil and Kolliphor EL and PEG 400 as a surfactant/co-surfactant mixture at
Using Triacetin as oil and Kolliphor EL and PEG 400 as a surfactant/co-surfactant mixture at
different ratios (Smix ), pseudo-ternary phase diagrams were constructed using the water titration
different ratios (Smix), pseudo-ternary phase diagrams were constructed using the water titration
method in order to obtain the concentration range of components necessary for preparing the NE.
method in order to obtain the concentration range of components necessary for preparing the NE.
After After
equilibrium waswas
equilibrium achieved,
achieved,each
each sample wasvisually
sample was visually checked
checked to determine
to determine if clearif NE
clear NE or
or an
an emulsion
emulsion waswaspresent.
present.The
Theobtained
obtained pseudo-ternary phasediagrams
pseudo-ternary phase diagrams prepared
prepared with
with a different
a different Smix Smix
with awith
fielda field
of existence of NE
of existence (non-shaded
of NE (non-shadedarea)
area)are
areshown in Figure
shown in Figure2.2.

FigureFigure 2. Pseudo-ternary
2. Pseudo-ternary phase
phase diagramsofofthe
diagrams thequaternary
quaternary systems
systems containing
containing Triacetin/Kolliphor
Triacetin/Kolliphor
EL/PEG 400/water at various Smix ratios. S1 : Smix (3:1), S2 : Smix (2:1), S3 : Smix S(1:1),
EL/PEG 400/water at various S mix ratios. S 1: S mix (3:1), S 2: Smix (2:1), S 3 : S mix (1:1), S4 : mix (1:2),
S4 :SS5:mix
Smix(1:2),
(1:3).
S5 : Smix (1:3).

Different
Different NE NE
areasareas
werewere obtainedas
obtained as the
the SSmix changed. The NE area reached its minimum at the
mix changed. The NE area reached its minimum at
highest co-surfactant concentration (Figure 2, S4 and S5 and a significant increase was observed as the
the highest co-surfactant concentration (Figure 2, S4 and S5 and a significant increase was observed
surfactant amount increased (Figure 2, S1, S2, and S3). Thus, an increasing surfactant amount relative
as the surfactant amount increased (Figure 2, S1 , S2 , and S3 ). Thus, an increasing surfactant amount
to the co-surfactant resulted in significant improvement of the NE area and the maximum was
relative to theatco-surfactant
reached resulted
a Smix of 2:1, while in significant
further SAA increase improvement of the NE
(Smix of 3:1) resulted in a area and the
narrower NE maximum
area. Many was
reached at a S
studies have of 2:1, while further SAA increase
mix shown that the NE area and position are (S of 3:1) resulted in a narrower NE
mixmainly determined by the applied Smix area. Many
ratio
studies have shown that the NE area and position are mainly determined by the applied
[40] and are affected by the applied oil type and concentration. For a specific oil/surfactant/co- S mix ratio [40]
and are affectedmixture,
surfactant by the applied oil type
the recorded NE and
area concentration.
in a phase diagram For aisspecific oil/surfactant/co-surfactant
an indicator of the emulsification
efficiency of the applied Smix. In this study, the maximum NE area at Smix of 2:1 was correlated with
Pharmaceutics 2019, 11, 40 9 of 18

mixture, the recorded NE area in a phase diagram is an indicator of the emulsification efficiency of the
applied Smix . In this study, the maximum NE area at Smix of 2:1 was correlated with the maximum
emulsification efficacy that resulted in the reduction of the interfacial tension to the minimal level and
optimum surfactant/co-surfactant packing with the formation of a highly flexible coherent film at the
oil/water interface [36].
Further investigation of the obtained phase diagrams shows that the detected NE region was
shifted toward the low-oil apex of the phase diagram, which confirms the importance of selecting
an oil of high drug solvation ability to ensure maximum drug loading.
NE formulations with a high SAA concentration are unfavorable since this may result in in vivo
irritation and also decreases the thermodynamic activity of the drug in the vehicle, which may affect
release of the drug from the applied dosage form [41]. Therefore, NE formulation systems capable of
attaining enhanced entropy and significantly lower oil/water interfacial free energy with the minimum
Smix were considered to be thermodynamically stable and were selected for further study.

3.3. Optimization of EPL-NE

Based on the constructed pseudo-ternary phase diagram, 20 plain NE formulations were prepared
and evaluated for droplet size, zeta potential, and PDI (data not shown). Only five plain NE systems
met the specified optimization criteria of droplet size in the range of 25 to 50 nm, zeta potential ranging
from −25 to −35 mV, a PDI less than 0.3, and extended transparency for one week at room temperature.
The composition of these NE formulations was as follows: At Smix of 3:1, Triacetin (10%), Kolliphor
EL (30%), PEG 400 (7%), and water (53%); and Triacetin (15%), Kolliphor EL (33%), PEG 400 (11%),
and water (41%). At Smix of 2:1; Triacetin (17%), Kolliphor EL (30%), PEG 400 (14%), and water (39%);
and Triacetin (24%), Kolliphor EL (30%), PEG 400 (17%), and water (28%). At Smix of 1:3, Triacetin
(5%), Kolliphor EL (12%), PEG 400 (36%), and water (47%). It was noted that higher oil concentrations
were obtained for NE systems prepared at Smix of 2:1, as indicated by maximum emulsification efficacy.
The optimized NE systems were selected for drug loading.

3.4. Characterization of EPL-NE Formulations

3.4.1. Drug Loading

The drug loading capacity of the prepared NE formulations varied from 37.91% (F5) to 99.11%
(F4) (Table 1), depending on oil content. The NE formulation with a higher oil content contained higher
amounts of the entrapped drug, which correlated with the higher solubility of the drug in Triacetin
(oil) and also confirms the importance of drug solubility as the main criterion for selection of oil.

3.4.2. Particle Size and Size Distribution

All prepared EP-NEs formulations had acceptable droplet sizes, ranging from 32.81 nm to
48.41 nm, with a narrow PDI, in the range of 0.160 to 0.240 (Table 1). PDI correlates the standard
deviation of the droplet size with the calculated mean size, which is a criterion for the uniformity of
size distribution within the NE formulation [20]. The small PDI values indicate that the systems are
mono-dispersed, have a narrow particle size distribution, and have a higher degree of stability [42].
Further investigation of the results showed that the oil content had a significant positive effect on
droplet size, which could be explained by the increased viscosity of the mixture due to increased
drug concentration. Surfactant and/or Smix did not have an effect on droplet size, mainly due to the
proximity of the applied SAA concentration in the optimized systems.

3.4.3. Zeta Potential

Zeta potential describes the electro-kinetic potential of colloidal systems and its value reflects
the intensity of electric charges at the oil/water interface. Zeta potential is the main parameter that
determines the physical stability of the dispersion, where repulsion between similar charges acts
Pharmaceutics 2019, 11, 40 10 of 18

against the Oswald ripening and prevents droplet aggregation with subsequent phase separation [43].
Zeta potential is controlled by the type and concentration of the applied SAA, system composition,
drug characteristics, vehicle nature, and presence of electrolytes. Generally, for the specific NE,
it was previously claimed that a zeta potential of ±30 mV indicates sufficient physical stability of
a system [20,44].
All optimized EPL-NE formulations had a zeta potential in the range of −25.2 to −35.7 mV
(Table 1), indicating good stability of the optimized systems.
The SAA affects zeta potential either directly through neutralization of surface charges or
indirectly through its effect on the particle size and surface area of the internal phase droplets [45].
In this study, the applied SAA and co-surfactant are nonionic and had a negligible effect on surface
charge; additionally, the proximity of SAA concentration in all optimized formulations downplays its
effect on zeta potential. The negative charge of the droplet surface mainly correlated with the fatty
acid content of the lipoid phase, with a slight difference between the NE systems according to their
lipid content and droplet size [19].

Table 1. Characterization of the prepared EPL- nanoemulsions (NE) formulations.

Code Smix Loading Capacity (%) Droplet Size (nm) Zeta Potential PDI
F1 67.42 ± 1.23 37.63 −27.3 0.196 ± 0.013
S1
F2 86.74 ± 1.46 42.99 −31.4 0.240 ± 0.053
F3 95.31 ± 1.91 43.74 −29.8 0.181 ± 0.099
S2
F4 99.11 ± 1.11 48.41 −35.7 0.160 ± 0.014
F5 S5 37.91 ± 1.09 32.81 −25.2 0.230 ± 0.017
PDI: Polydispersity index.

3.5. EPL-NE Liquisolids

The liquisolid technique is an advanced approach used mainly for enhancing the water solubility
of a drug by mixing it with a suitable water miscible vehicle and loading onto a powder mixture
of high absorbent and/or adsorbent properties (carrier and coat) in specified weights calculated
according to the proposed mathematical model to achieve complete dryness and ensure good flow
and compressibility characteristics [30]. In other words, liquisolid compacts could be described as
liquid medications with an adequate flow that are compressed into a powdered form. In this work,
a liquisolid mixture was applied as a carrier for the prepared EPL-NE formulations with good flow
characteristics and that was suitable for direct compression using Avicel/nano-sized silicone dioxide as
a carrier/coat mixture; thus, no water miscible vehicle was added and the calculations were dependent
on the weight of the dry EPL-NE formulation that was loaded as a liquid medication.
According to the mathematical model, the flowable liquid retention potentials (Φ-Value) of Avicel
and nano-sized silicone dioxide (R = 20) were used to calculate the liquid load factor (Lf ); the latter
was used to calculate the amounts of carrier and coat materials that depended on the weight of the
applied dry EPL-NE formulation according to the following equation:

Lf = W/Q (1)

where W is the weight of the dry EPL-NE formulation and Q is the weight of the carrier. The amount
of coating material was calculated from the R-value. The composition and flow characterization of the
prepared EPL-NE liquisolids are shown in Table 2.
Highly flowable characteristics are essential for mixtures that are prepared for direct compression
to guarantee homogenous mixing, efficient die fill, uniform tablet weight, precise drug content,
and ultimately accepted therapeutic value [15]. The flow characteristics of the prepared EPL-NE
liquisolids were assayed by measuring the angle of repose, Carr’s index, and Hausner ratio.
Pharmaceutics 2019, 11, 40 11 of 18

Based on the results in Table 2, all prepared EPL-NE liquisolid mixtures showed very good
flow characteristics, with an angle of repose in the range of 27.61◦ to 32.41◦ , Carr’s index values less
than 20 (in the range of 16.02 to 19.37%), and the Hausner ratio close to unity [24]. These results
designate the precision of the applied calculations of different model parameters and indicate the
suitability of selecting a liquisolid technique as a carrier for the prepared EPL-NE formulation for
direct compression.

Table 2. Composition and pre-compression characterization of EPL-NE liquisolid formulations.

EPL Hausner
F Lf Q q Disg. W (mg) θ Ci%
(mg) Ratio
F1 223.8 11.9 23.6 291.7 28.75 16.92 1.17
F2 280.9 14.1 29.5 352.4 31.82 18.11 1.16
F3 25 0.21 290.5 14.5 30.5 363.5 32.41 19.37 1.13
F4 338.1 16.9 35.5 419.1 30.82 17.41 1.15
F5 252.4 12.6 26.5 319.2 27.61 16.02 1.07
F: Formulation. Lf : Liquid load factor. Q: Carrier weight. q: Coat weight. θ: Angle of repose. Ci%: Carr’s index.
Disg.: Disintegrant weight. W: Unit dose weight.

3.6. Characterization of EPL-NE Liquisolid Tablets

Characterization results of the prepared EPL-NE liquisolid tablets are shown in Table 3.
All formulations showed a small weight variation and high drug content, ranging from 96.12%
to 100.15%, which, according to standardized pharmacopeial limits, correlates with and indicates
that the prepared liquisolid formulation mixtures had good flow characteristics. Hardness values
above 50 N and friability less than 1% indicate good mechanical properties and adequate breaking
strength, which can be correlated with the high compressibility characteristics of the formulation
components (Avicel and Pharmaburst). Selection of Pharmaburst-500 SPI for use as a superdisintegrant
for liquisolid tablet formulations not only enhanced the mechanical properties but also had a positive
effect on disintegration. Pharmaburst is a co-processed mixture of mannitol, starch, crosspovidone,
crosscarmellose-Na, and silica [46]. The combined wicking and swelling properties of both carmellose
and povidone resulted in rapid tablet disintegration within 41 to 53 seconds for formulations F1 and
F4, respectively.

Table 3. Post-compression characterization of EPL-NE liquisolid tablets.

Average Weight Drug Content Thickness Diameter Friability Hardness Dis. Time
F
(mg) ± S.D. (%) ± S.D. (cm) (cm) (%) (N) (Seconds)
F1 289.12 ± 3.6 97.89 ± 2.1 0.15 0.8 0.5 55 41
F2 349.7 ± 2.9 99.19 ± 1.3 0.21 0.8 0.4 56 45
F3 365.2 ± 3.1 100.15 ± 0.8 0.21 0.8 0.4 59 47
F4 422.1 ± 3.2 100.08 ± 0.2 0.23 0.8 0.3 63 53
F5 317.9 ± 2.9 96.12 ± 3.4 0.18 0.8 0.6 55 44
N: Newton. Dis.: Disintegration.

3.7. Drug Release Study

Figure 3 shows the dissolution profiles of EPL from different prepared EPL-NE liquisolid tablet
formulations in comparison to EPL-NEs and unformulated EPL in 0.1 N HCl (pH 1.2). The percent drug
release showed a remarkable increase from all liquisolid formulations, in which approximately 90% of
the drug was released after 45 minutes and drug release was complete within one hour, as compared
to 39.6% and 47.3% for the unformulated drug after 45 and 60 minutes, respectively. For EPL-NE,
a significant improvement of drug release was observed but with a sustained pattern, in which nearly
complete drug release (greater than 97%) occurred within four hours. Further investigation of the
release data indicated that the drug release profiles of both prepared EPL-NEs and EPL-NE liquisolids
Pharmaceutics 2019, 11, 40 12 of 18

showed a biphasic release pattern, with rapid initial drug release in which 60–70% of the drug dose was
released within one hour and 15 minutes for EPL-NEs and EPL-NE liquisolids, respectively. This could
be explained by the higher wettability of the drug in contact with water due to the SAA effect that is
Pharmaceutics 2018, 10, x FOR PEER REVIEW 12 of 18
favored in liquisolids by the presence of the finely-sized particle component of liquisolid (nano-silica)
that acts
water asdue
a distributing
to the SAA effect agentthatvia the adsorption
is favored effect
in liquisolids byon
theapresence
larger surface area and particle
of the finely-sized rapid drug
partitioning
component intoofdiluted
liquisolid dissolution
(nano-silica) medium,
that acts mainly from small
as a distributing agentdroplets. A slowereffect
via the adsorption drugonrelease
a
patternlarger surface
in the secondareaphase
and rapid
coulddrugbe partitioning
correlated with into diluted dissolution
the decline medium,drug
in oil/water mainly from smallfrom
partitioning
largerdroplets.
dropletsAunder slower drug
the release
effect pattern inmedia
of increasing the second
drugphase could be correlated
concentration accordingwith to thethe decline in
Noyes-Whitney
equation [47]. The effect of droplet size distribution (PDI) on drug release from nanoemulsiondrug
oil/water drug partitioning from larger droplets under the effect of increasing media systems
concentration according to the Noyes-Whitney equation [47]. The effect of droplet size distribution
was verified by Ritger and Peppas [48] and confirmed by many researchers [49]. Our results are in
(PDI) on drug release from nanoemulsion systems was verified by Ritger and Peppas [48] and
agreement with many studies that concluded that conventional NE formulations usually result in
confirmed by many researchers [49]. Our results are in agreement with many studies that concluded
enhancement of hydrophobic
that conventional drug dissolution
NE formulations usually result butinwith a sustained
enhancement release pattern
of hydrophobic drugunder the effect
dissolution
of oil but
andwith
interfacial
a sustained release pattern under the effect of oil and interfacial film barriers [50]. However, of
film barriers [50]. However, in this work, it was clearly seen that the loading
the NE in formulation
this work, it wasontoclearly
a liquisolid mixture
seen that resulted
the loading in significant
of the NE formulationenhancement of both mixture
onto a liquisolid the amount
released and the
resulted rate. Thisenhancement
in significant could be correlated
of both the toamount
the factreleased
that evenandthough
the rate.the
Thisdrug
couldprepared in a solid
be correlated
to the fact that even though the drug prepared in a solid dosage form,
dosage form, it is detained within the formulation powder blend in solution or in an almost molecularlyit is detained within the
formulation powder blend in solution or in an almost molecularly dispersed
dispersed form, which causes a significant increase in surface area that is available for dissolution form, which causes a and
significant increase in surface area that is available for dissolution and the
the degree of subdivision of the drug loaded oil droplets with improved wetting characteristics [25]. degree of subdivision of
the drug loaded oil droplets with improved wetting characteristics [25]. This effect on the drug release
This effect on the drug release profile is highly advantageous since it allows faster drug absorption
profile is highly advantageous since it allows faster drug absorption to therapeutic levels in addition
to therapeutic levels in addition to the overall increase in drug absorption, which mainly occurs in
to the overall increase in drug absorption, which mainly occurs in gastric and upper duodenal
gastric and upper
segments duodenal segments [12,14].
[12,14].

Figure
Figure 3. Release
3. Release profiles
profiles of EPL
of EPL from
from (a):NE-Liquisolids
(a): NE-Liquisolids and
and(b):
(b):NE
NEformulations in comparison
formulations to to
in comparison
unformulated
unformulated EPL.EPL.

EPL release
EPL release datadata
fromfrom
thethe EPL-NEsand
EPL-NEs andEPL-NE
EPL-NE liquisolids
liquisolidswas
wasfitted to different
fitted kinetic
to different models
kinetic models
and then the best fit was selected based on the calculated correlation coefficient
and then the best fit was selected based on the calculated correlation coefficient (R ) valueeach (R 2) value
2 for for each
model. The Weibull model showed the best fit with an R2 value that exceeded 0.9994 for the EPL-NE.
model. The Weibull model showed the best fit with an R2 value that exceeded 0.9994 for the EPL-NE.
For EPL-NE liquisolids, drug release followed the Korsemeyer-Peppas model with R2 values greater
For EPL-NE liquisolids, drug release followed the Korsemeyer-Peppas model with R2 values greater
than 0.9986. These results indicate uniformity of drug solubility and distribution within the lipid
than 0.9986.
phase, withThese results indicate
a subsequent complexuniformity of drug solubility
mixed dissolution-diffusion andpattern
release distribution within theinlipid
that is predictable
phase,NEwith a subsequent
systems complex
and typically mixedbydissolution-diffusion
described the Weibull model. Forrelease pattern that is predictable
the Korsemeyer-Peppas model, ain NE
systems and typically described by the Weibull model. For the Korsemeyer-Peppas
number of synchronized steps usually occur that are expected for modified tablets dosage forms model, a number
of synchronized steps usually
including diffusion of water,occur
tablet that are with
swelling expected for modified
subsequent tabletsand
drug diffusion, dosage forms These
dissolution. including
results
diffusion ofclearly
water,demonstrate the effect
tablet swelling withof subsequent
liquisolid loading
drugondiffusion,
drug releaseandfrom EPL-NE formulations
dissolution. These results
[51].
clearly demonstrate the effect of liquisolid loading on drug release from EPL-NE formulations [51].
Based on previous in vitro characterization results, EPL-NE liquisolid formulation No. 4 (F4)
was selected and subjected to further solid-state characterizations and bioavailability studies.
Pharmaceutics 2019, 11, 40 13 of 18

Based on previous in vitro characterization results, EPL-NE liquisolid formulation No. 4 (F4) was
selected and
Pharmaceutics subjected
2018, 10, x FORtoPEER
further solid-state characterizations and bioavailability studies.
REVIEW 13 of 18

3.8. Solid State

3.8. Solid State Characterizations
Characterizations and
and Compatibility
Compatibility Studies
Studies
Solid state
statecharacterizations
characterizationsand and compatibility
compatibility studies
studies were
were performed
performed withwith the optimized
the optimized EPL-
EPL-NE liquisolid formulation (F4) in comparison to formulation
NE liquisolid formulation (F4) in comparison to formulation physical mixture and unformulatedphysical mixture and
unformulated
drug. drug.
DSC is a reliable and simple tool to test for materialmaterial purity,
purity, formulation
formulation mixture
mixture compatibility,
compatibility,
and describing drug crystalline properties in a specific blend, which is affected by drug solubility
and/or distribution state
and/or distribution state within
within the
the mixture.
mixture. Figure
Figure 4a4a shows
shows the DSC thermogram of unformulated
EPL with a sharp endothermic melting peak at 231.13 ◦ C in accordance with previously published
sharp endothermic 231.13°C previously
data [12], indicating drug purity, which which completely
completely disappeared in thermograms of the liquisolid
formulation under the effect of drug solubility within the oil core and loss of crystallinity. The drug
melting peak was retained in the thermogram of the physical mixture without the appearance of any
new peaks
peaksor or
other thermal
other events,events,
thermal indicating compatibility
indicating between formulation
compatibility mixture components.
between formulation mixture
Figure 4b shows the IR spectrum of unformulated EPL with the main characteristic
components. Figure 4b shows the IR spectrum of unformulated EPL with the main characteristic functional groups
at 2988.65 cm − 1 (C–H stretching), − 1 − 1
functional groups at 2988.65 cm 1778.08
−1 cm (anhydride
(C–H stretching), 1778.08 O–C=O stretching), O–C=O
cm (anhydride
−1 1726.22 cm (C=O
stretching),
ester stretching),
1726.22 cm−1 (C=O andester
(1657.64 cm−1 ) C=O
stretching), andstretching.
(1657.64 IR cmspectra
−1) C=O of stretching.
EPL-NE liquisolid and the
IR spectra of physical
EPL-NE
mixture
liquisolidshowed
and the nophysical
chemicalmixture
changesshowed
where allnodrug functional
chemical groups
changes wereall
where retained, which confirmed
drug functional groups
the
werecompatibility of EPL
retained, which with formulation
confirmed additives.
the compatibility of EPL with formulation additives.

Figure 4. Solid state characterization of EPL-NE liquisolid formulation using (a) differential scanning
Figure 4. Solid state characterization of EPL-NE liquisolid formulation using (a) differential scanning
calorimetry (DSC) and (b) FTIR.
calorimetry (DSC) and (b) FTIR.
3.9. In Vivo Characterization Studies
3.9. In Vivo Characterization Studies
To study the effect of increased solubility and the improved release rate of EPL from the prepared
To studyformulation
NE liquisolid the effect of
on increased solubility
drug absorption and and the improved
bioavailability, release rate
the optimized of EPL from
formulation the
(F4) was
prepared NE liquisolid formulation on drug absorption and bioavailability, the optimized
compared to the corresponding EPL-NE and conventional oral tablets in rabbit bioavailability studies.
formulation (F4) was compared to the corresponding EPL-NE and conventional oral tablets in rabbit
bioavailability studies. The mean pharmacokinetic parameters of EPL of different tested formulations
are summarized in Table 4 and the mean plasma concentration-time profiles are illustrated in Figure
5.

Table 4. Mean pharmacokinetic parameters of EPL after administration to rabbits in different forms.
Pharmaceutics 2019, 11, 40 14 of 18

The mean pharmacokinetic parameters of EPL of different tested formulations are summarized in
Pharmaceutics 2018, 10, x FOR PEER REVIEW 14 of 18
Table 4 and the mean plasma concentration-time profiles are illustrated in Figure 5.

Table Pharmacokinetic EPL-NEof EPL after administration to rabbits in different forms.

4. Mean pharmacokinetic parameters EPL-NE EPL Tablets
parameter Liquisolids
Pharmacokinetic
Cmax (ng/ml)Parameter EPL-NE± Liquisolids
1986.76 53.6 1505.51EPL-NE
± 24.91 EPL± Tablets
946.34 46.27
CT (ng/mL)
max (hr)
max 1986.76
0.95 ± 0.048± 53.6 1505.51
1.04 ± 24.91
± 0.023 946.34 ± 46.27
1.38 ± 0.021
Tmax (hr) 0.95 ± 0.048 1.04 ± 0.023 1.38 ± 0.021
KK(hr(hr−1))
−1 0.1870.187
± 0.031
± 0.031 0.164 ± 0.042
0.164 ± 0.042 0.196 ± 0.028
0.196 ± 0.028
t1/2
t1/2(hr)
(hr) 3.7193.719
± 0.12
± 0.12 4.238 ± 0.18
4.238 ± 0.18 3.547 ± 0.07
3.547 ± 0.07
AUC(0–4)
AUC (ng·hr/mL)
(0–4)(ng·hr/ml) 11178.56
11178.56 ± 74.47
± 74.47 9108.76
9108.76 ± 35.27
± 35.27 5293.15
5293.15 ± 27.33
± 27.33
AUC (0–α) (ng·hr/mL) 11328.11 ± 35.78 9292.19 ± 23.38 5383.56 ± 18.72
AUC (0–α) (ng·hr/ml) 11328.11 ± 35.78 9292.19 ± 23.38 5383.56 ± 18.72

Bioavailability
Bioavailability is is generally
generally defined
defined as as the
the rate
rate and
and extent
extent of
of drug
drug absorption.
absorption. In In bioavailability
bioavailability
calculations
calculations ofof plasma
plasma data,
data, the
the extent
extent ofof absorption
absorption is is usually
usually quantified
quantified and and described
described byby both
both area
area
under plasma concentration time curve (AUC) and the drug maximum
under plasma concentration time curve (AUC) and the drug maximum plasma concentration (Cmax plasma concentration (C max))
while time elapsed to reach maximum plasma
while time elapsed to reach maximum plasma concentration (Tmax concentration (T ) is the parameter that describes
max) is the parameter that describes
absorption
absorption rate [15]. In this study, major changes in all pharmacokinetic parameters
rate [15]. In this study, major changes in all pharmacokinetic parameters werewere observed,
observed,
indicating an improvement in both the rate and extent of
indicating an improvement in both the rate and extent of drug absorption. Fordrug absorption. For liquisolid formulations,
liquisolid
the mean Cmaxthe
formulations, and AUCC0–α
mean wereAUC
max and increased
0- α wereand reached and
increased 1986.76 ng/mL
reached and 11,328.11
1986.76 ng/ml andng.hr/mL,
11,328.11
respectively, as compared to 1505.51 ng/mL and 9292.19 ng.hr/mL
ng.hr/ml, respectively, as compared to 1505.51 ng/ml and 9292.19 ng.hr/ml for the EPL-NE for the EPL-NE formulation,
while lower plasma
formulation, while concentrations
lower plasma were observed for
concentrations conventional
were observed oral tablets, with Coral
for conventional max and AUCwith
tablets, 0–α
values
Cmax andof AUC
946.340–α ng/mL
values of and 5383.56
946.34 ng/mlng·and
hr/mL, respectively.
5383.56 ng·hr/ml, The results also
respectively. Theshowed a shortened
results also showed
Tamax of 0.95 hours
shortened Tmax ofand 0.951.04 hours
hours andfor1.04
liquisolid
hours forandliquisolid
NE formulations, respectively, as
and NE formulations, compared to
respectively, as
1.38 hours for conventional tablet formulation. As dissolution is the rate
compared to 1.38 hours for conventional tablet formulation. As dissolution is the rate determining determining step in the
absorption process of process
step in the absorption BCS class of II
BCSdrugs,
classthese results
II drugs, these correlate with the significant
results correlate increase increase
with the significant in drug
dissolution
in drug dissolution from liquisolid formulations, the positive effect of NE on permeation throughGIT
from liquisolid formulations, the positive effect of NE on permeation through the the
lumen, and para-cellular
GIT lumen, absorption
and para-cellular that allows
absorption thatthe drug the
allows to escape
drug liver metabolism,
to escape with subsequent
liver metabolism, with
higher plasma
subsequent levels.
higher plasma levels.

Figure Mean plasma

Figure 5.5. Mean plasma concentration
concentration time
time curves
curves of
of EPL
EPL from
from different
different tested
tested forms
forms after
after
administration
administration to
to rabbits.
rabbits.

Statistical analysis of
Statistical analysis ofthe
thecalculated
calculatedpharmacokinetic
pharmacokinetic parameters
parameters is shown
is shown in Tables
in Tables 5 6,
5 and and 6,
with
with
95% 95% confidence
confidence intervals.
intervals. According
According to these
to these results,
results, a significantdifference
a significant differencewas
was observed
observed at
at the
the
selected probability level. Additional data analysis was performed using the post-hoc tukey model
to pinpoint the exact position of the observed difference, which indicated a significant difference in
both the rate and extent of drug absorption for EPL-NE liquisolid as compared to conventional tablets
but the only significant difference in the extent of absorption that was observed was in comparison
Pharmaceutics 2019, 11, 40 15 of 18

selected probability level. Additional data analysis was performed using the post-hoc tukey model to
pinpoint the exact position of the observed difference, which indicated a significant difference in both
the rate and extent of drug absorption for EPL-NE liquisolid as compared to conventional tablets but
the only significant difference in the extent of absorption that was observed was in comparison with the
EPL-NE formulation. A significant difference in both the rate and extent of drug absorption between
conventional tablets and EPL-NE formulation was observed. The 95% confidence intervals had narrow
ranges and excluded zero, which indicates data accuracy, the strength of the results, and confirmed the
rejection of the null hypothesis.

Table 5. One way ANOVA test for EPL pharmacokinetic data from different forms in rabbits.

Sum of
Parameter Source df Mean Square F Sig.
Squares
Between Groups 3,131,899.76 2 1,565,949.88
Cmax (ng/mL) Within Groups 5123.96 15 4584.19
341.597
Total 3,137,023.72 17
Between Groups 0.444 2 0.222
Tmax (hr) Within Groups 0.198 15 16.84
0.013
Total 0.642 17
<0.001
Between Groups 106,305,369.09 2 53,152,684.55
AUC (0–24) Within Groups 2,788,214.49 15 285.95
(ng·hr/mL) 185,880.97
Total 109,093,583.59 17
Between Groups 108,822,905.28 2 54,411,452.64
AUC (0–α) Within Groups 3,195,313.44 15 255.43
(ng·hr/mL) 213,020.89
Total 112,018,218.72 17
Between Groups 0.001 2 0.001
K (hr−1 ) Within Groups 0.001 15 9.38 0.002
0.000
Total 0.003 17
Between Groups 0.804 2 0.402
t1/2 (hr) Within Groups 0.679 15 8.88 0.003
0.045
Total 1.482 17
df: degree of freedom.

Table 6. Post-Hoc statistical analysis of the calculated means with 95% confidence intervals.

Confidence Interval
Parameter Group Mean Difference
Lower Upper
EPL Tablets 1020.468 * 992.751 1048.185
Cmax (ng/mL)
EPL-NE 465.968 * 438.251 493.685
EPL Tablets −0.334 * −0.506 −0.161
Tmax (hr)
EPL-NE Liquisolids

EPL-NE 0.0011 0.0007 0.012

EPL Tablets 0.0059 0.003 0.019
K (hr−1 )
EPL-NE 0.0214 * 0.008 0.035
EPL Tablets −0.129 −0.4481 −0.120
t1/2 (hr)
EPL-NE −0.499 * −0.8176 −0.179
EPL Tablets 5864.821 * 5218.263 6511.379
AUC (0–24) (ng·hr/mL)
EPL-NE 2049.661 * 1403.103 2696.218
EPL Tablets 5924.305 * 5232.155 6616.457
AUC (0–α) (ng·hr/mL)
EPL-NE 2022.642 * 1330.491 2714.793
*: Significant model term.
Pharmaceutics 2019, 11, 40 16 of 18

4. Conclusions
NEs are lipid formulations that can easily pass through the GIT membranes and bypass liver
degradation due to lymphatic absorption, which significantly enhances the bioavailability of poorly
soluble drugs, especially those that suffer liver metabolism. In this work, EPL as a BCS class II drug
model that normally suffers extensive liver degradation was formulated into an NE using Triacetin (oil)
and Kolliphor EL/PEG 400 (Smix ) in different ratios. Optimized NE formulations were loaded onto free
flowing, highly compressible liquisolid tablet mixtures for oral delivery and the drug bioavailability of
these NE formulations were compared to conventional oral tablets and NE formulation. Drug release
from optimized EPL-NE liquisolids was significantly increased, with a high rate that exceeded 90%
within 45 minutes, while EPL-NE also showed significant drug release but with a sustained pattern
over four hours. The rate and extent of drug absorption were significantly increased from liquisolids
as compared to NE formulations and conventional tablets, as indicated by the AUC, Cmax , and Tmax ,
in which bioavailability from liquisolids reached 2.1 relative to conventional tablets and 1.21 relative
to the NE formulation. The bioavailability of EPL from NE formulation was increased by 1.72 relative
to conventional oral tablets.
These results support the conclusion that the EPL-NE liquisolid mixture is a promising dosage
form for oral delivery with good flow and compression characteristics that increase the release
rate, enhance drug absorption, and allows the drug to escape liver degradation, with a subsequent
significant increase in drug bioavailability. In addition, Nanoemulsion Liquisolids, are expected to
be a promising oral delivery system for BCS class II drugs with poor bioavailability especially those
liable to extensive liver degradation. Further studies on other drug models in terms of pK, logP, and
permeability are required to confirm the suitability of nanoemulsion liquisolids as an oral delivery
system to increase drug bioavailability and protect from liver degradation.

Funding: This work received no external funding.

Conflicts of Interest: The author of the article declares no conflict of interests in relation to this study and no
financial support was received in the preparation of this work.

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