Enhancement of oral bioavailability of atorvastatin calcium by self-emulsifying drug delivery systems (SEDDS)

Abstract

The aim of the present study was to formulate a self-emulsifying drug delivery system of atorvastatin calcium and its characterization including in vitro and in vivo potential. The solubility of atorvastatin calcium was determined in various vehicles such as Captex 355, Captex 355 EP/NF, Ethyl oleate, Capmul MCM, Capmul PG-8, Gelucire 44/14, Tween 80, Tween 20, and PEG 400. Pseudoternary phase diagrams were plotted on the basis of solubility data of drug in various components to evaluate the microemulsification region. Formulation development and screening was carried out based on results obtained from phase diagrams and characteristics of resultant microemulsion. Prepared formulations were tested for microemulsifying properties and evaluated for clarity, precipitation, viscosity determination, drug content and in vitro dissolution. The optimized formulation further evaluated for particle size distribution, zeta potential, stability studies and in vivo potential. In vivo performance of the optimized formulation was evaluated using a Triton-induced hypercholesterolemia model in male Albino Wistar rats. The formulation significantly reduced serum lipid levels as compared with atorvastatin calcium. Thus studies illustrated the potential use for the delivery of hydrophobic drug such as atorvastatin calcium by oral route.

Full text

Introduction
Approximately 40% of new drug candidates are
hampered for further development because of their
high lipophilicity and poor water solubility and thus
leads to low bioavailability, high intra and inter-subject
variability and a lack of dose proportionality.[1] Drug
discovery strategies based on the technology of com-
binatorial chemistry information, high throughput
screening, genomics, robotic technology and mini-
aturization have increased the drug libraries of many
pharmaceutical companies to millions. However, during
drug discovery process attempts which are being made
to enhance pharmacological activities of New Chemical
Entity (NCE), very basic physico-chemical properties like
solubility always suer a great deal. Drug molecules with
restricted aqueous solubility are becoming more and
more common in the research and development port-
folios of discovery- focused pharmaceutical companies.
To triumph over these problems, various formulation
strategies are exploited such as use of surfactants, lipids,
permeation enhancers, micronization, salt formation,
cyclodextrins, nanoparticles and solid dispersions. Each
and every method for bioavailability enhancement has
its own merits and demerits. Recently, much attention
has been paid to lipid-based formulations with particu-
lar emphasis on self-emulsifying drug delivery systems
(SEDDS) to improve the oral bioavailability of lipophilic
drugs.[2] SEDDS are dened as an isotropic mixture
of natural or synthetic oils, solid or liquid surfactants
or alternatively one or more hydrophilic solvents and
co-solvents/surfactants. Upon peroral administration,
Pharmaceutical Development and Technology
Pharmaceutical Development and Technology, 2011; 16(1): 65–74
2011
16
1
65
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Address for Correspondence: Dr Surendra G. Gattani, Prof. and Head, Department of Pharmaceutics and Quality assurance, R. C. Patel Institute of
Pharmaceutical Education and Research, Near Karwand Naka, Shirpur 425 405, Dhule, Maharashtra, India. Tel: +91 099708 16927. Fax: +91 02563 255189.
Email: sggattani@redimail.com
21 March 2009
13 November 2009
13 November 2009
1083-7450
1097-9867
© 2011 Informa Healthcare USA, Inc.
10.3109/10837450903499333
RESEARCH ARTICLE
Enhancement of oral bioavailability of atorvastatin
calcium by self-emulsifying drug delivery systems
(SEDDS)
Pawan J. Kadu, Sachin S. Kushare, Dhaval D. acker, and Surendra G. Gattani
R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dhule, Maharashtra, India
Abstract
The aim of the present study was to formulate a self-emulsifying drug delivery system of atorvastatin calcium
and its characterization including in vitro and in vivo potential. The solubility of atorvastatin calcium was
determined in various vehicles such as Captex 355, Captex 355 EP/NF, Ethyl oleate, Capmul MCM, Capmul
PG-8, Gelucire 44/14, Tween 80, Tween 20, and PEG 400. Pseudoternary phase diagrams were plotted on
the basis of solubility data of drug in various components to evaluate the microemulsification region.
Formulation development and screening was carried out based on results obtained from phase diagrams
and characteristics of resultant microemulsion. Prepared formulations were tested for microemulsifying
properties and evaluated for clarity, precipitation, viscosity determination, drug content and in vitro dis-
solution. The optimized formulation further evaluated for particle size distribution, zeta potential, stability
studies and in vivo potential. In vivo performance of the optimized formulation was evaluated using a
Triton-induced hypercholesterolemia model in male Albino Wistar rats. The formulation significantly reduced
serum lipid levels as compared with atorvastatin calcium. Thus studies illustrated the potential use for the
delivery of hydrophobic drug such as atorvastatin calcium by oral route.
Keywords: Atorvastatin calcium; self emulsifying drug delivery system; bioavailability enhancement;
Triton-induced hypercholesterolemic in vivo evaluation
PHD
450351
(Received 21 March 2009; revised 13 November 2009; accepted 13 November 2009)
ISSN 1083-7450 print/ISSN 1097-9867 online © 2011 Informa Healthcare USA, Inc.
DOI: 10.3109/10837450903499333 http://www.informahealthcare.com/phd
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66 P.J. Kadu et al
these systems shape ne oil-in-water emulsions (or
microemulsions) in the gastrointestinal (GI) tract with
mild agitations provided by gastric mobility. ese
systems advantageously present the drug in dissolved
form and the small droplet size provides a large inter-
facial area for solvent drug interaction and ultimately
drug absorption.[1,3] For eective in vivo performance of
SEDDS, the formulation should form droplets of size <
5 µ. e smaller oil droplets oer a large interfacial area
for pancreatic lipase to hydrolyze triglycerides and thus
promote the rapid release of the drug.[4] Selection of a
suitable self emulsifying formulation depends upon the
assessment of solubility of the drug in various compo-
nent, area of the self-emulsifying region as obtained in
the phase diagram, and droplet size distribution of the
resultant emulsion following self-emulsication.[4,5]
Atorvastatin calcium (ATR) is a selective and com-
petitive inhibitor of hydroxyl methyl glutaryl-coenzyme
A (HMG-CoA) reductase. is enzyme is responsible
for converting HMG-CoA to mevalonate, which is a
precursor of cholesterol synthesis. As a result of inhibi-
tion of the enzyme by ATR there occurs a decrease in
mevalonate level, and a subsequent decrease in hepatic
cholesterol levels and increase in uptake of low density
lipoprotein cholesterol (LDL-CH). us ATR is used
to lower cholesterol and triglycerides in patients with
hypercholesterolemia and mixed dyslipidemia and in the
treatment of homozygous familial hypercholesterolemia.
ATR has a unique structure, long half-life, and hepatic
selectivity, explain its greater LDL-CH lowering potency
compared to other HMG-CoA reductase inhibitors.
ATR-[R-(R*,R*)]-2-(4-uorophenyl)- b,d-dihydroxy-5-
(1-methylethyl)-3-phenyl-4-[(phenyl amino) carbonyl]-
1H-pyrrole-1-heptanoic acid, calcium salt (2:1) trihydrate,
is a white to o-white crystalline powder that is insoluble
in aqueous solutions of pH 4 and below, which are the
conditions typically present in the stomach of a subject.
ATR is very slightly soluble in distilled water, pH 7.4
phosphate buer and acetonitrile, slightly soluble in etha-
nol, and freely soluble in methanol, also rapidly absorbed
after oral administration. e absolute bioavailability of
ATR is approximately 14% and as per Biopharmaceutical
Classication System; the drug is classied as a class
II drug.[6–8] For such drugs, dissolution in GI lumen is
a rate controlling step for absorption and subsequent
therapeutic action. Improved absorption can be achieved
by the use of delivery systems, which can enhance drug
dissolution from its dosage form and maintains drug
in dissolved state in GI uids. erefore, the current
scenario demands a need for a delivery strategy that can
improve its therapeutic ecacy. SEDDS recently have
gained great interest in drug delivery research for their
potential in improving oral bioavailability of poorly water
soluble drugs as compared to conventional approaches.
e main objective of the study was to develop and
evaluate an optimal SEDDS formulation containing ATR
and to assess its pharmacodynamic eect in terms of lipid
lowering potential.
Materials and methods
Materials
ATR was a generous gift from FDC Ltd, India. Triglycerides
of caprylic/capric acids (Captex® 355), Triglycerides of
caprylic/capric acids (Captex® 355 EP/NF), C8/C10
mono-/diglycerides (Capmul® MCM) and propylene glycol
caprylate (Capmul PG-8), were generous gifts from Abitec
Corporation USA. Gelucire 44/14 was received as a gift
from Colorcon Asia Pvt. Ltd, Goa, India. Polyethylene
glycol 400 was purchased from Merck Specialties Pvt.
Ltd, Mumbai. Tween 80, Tween 20 and Ethyl oleate
LR were purchased from SD Fine Chem. Ltd, Mumbai.
Empty hard gelatin capsules were obtained as gift from
Associated Capsules Pvt. Ltd, Mumbai. Triton WR 1339
and Sigma® Dialysis Tubing (seamless cellulose tubing,
MWCO 12000) was purchased from Sigma Chemical Co,
USA. In vitro diagnostic kits (Cholesterol LS, LDL-CH
and Triglycerides) were purchased from RFCL Pvt. Ltd,
Haridwar, India. All other chemicals and reagents used
were of analytical grades.
Methods
Solubility studies
e objective of solubility is to determine the solubiliza-
tion capacity for drug in given vehicles. Vehicles which
show highest solubility are then used for formulation
of SEDDS. e solubility of ATR in various vehicles, i.e.
oils (Captex 355, Captex 355 EP/NF, Ethyl oleate LR),
surfactants (Capmul MCM, Capmul PG-8, Gelucire
44/14, Tween 80, Tween 20) and cosurfactant (PEG 400)
was determined initially. A total of 5 mL of each of the
selected vehicles were added to each cap vial containing
an excess of ATR. Vials were then shaken for 48 h in an
orbital shaker (Remi Instruments, Mumbai, India) main-
tained at 37 ± 1°C. After reaching equilibrium, each vial
was centrifuged at 3000 g for 5 min, and excess insoluble
ATR was discarded by ltration using Whatman lter
paper (No. 41). e concentration of solubilized ATR
was quantied by UV spectrophotometer at 241 nm
(Shimadzu-1700, Japan).[5]
Pseudoternary phase diagram study
Pseudoternary phase diagrams of oil, surfactants/
cosurfactant or cosolvents (S/CoS), and water were
developed using the water titration method. e mixtures
of oil and S/CoS at certain weight ratios were diluted with
water in a drop wise manner. For each phase diagram at
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Enhancement of oral bioavailability of atorvastatin calcium 67
a specic ratio of surfactants, i.e. (1:1, 2:1) and S/CoS (i.e.
1:1, 2:1, 3:1, 4:1 and 5:1 wt/wt), a transparent and homog-
enous mixture of oil and S/CoS was formed. en, each
mixture was titrated with water and visually observed for
phase clarity and ow ability.[5] After the identication of
microemulsion region in the phase diagrams, the micro-
emulsion formulations were selected at desired compo-
nent ratios. In order to form the stable microemulsion, a
series of SEDDS formulations were prepared (Figure 2).
Preparation of SEDDS formulations
A series of SEDDS formulations were prepared using
Captex 355 as the oil, Capmul MCM and Tween 80 as
surfactants mixture (Smix), and PEG 400 as cosolvent
(Table 1). In all the formulations, the level of ATR was
kept constant (i.e. 1.9% wt/wt of the total formulation
weight). Accurately weighed ATR was placed in a glass
vial, and surfactant (Capmul MCM) was added and mixed
by gentle stirring on a magnetic stirrer at 40°C until ATR
was completely dissolved. e remaining components,
i.e. Tween 80, Captex 355 and PEG 400 were added with
constant stirring at 40°C until stable mixture was formed.
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10
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Tween 20
Ethyle oleate
Captex 355
Captex 355 NF
Capmul MCM
Gelucire 44/14
PEG400
Components
Figure 1. Solubility of ATR in various components.
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Smix 1:1, S/Cos 2:1 Smix 1:1, S/Cos 3:1
Register CHEMIX Now!
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Figure 2. (a) Pseudoternary phase diagrams of oil, water and S/Cos ratio for Smix 1:1. (b) Pseudoternary phase diagrams of oil, water and S/Cos
ratio for Smix 2:1.
Figure 2. continued on next page
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68 P.J. Kadu et al
e mixture was stored at room temperature until further
use.[5]
Characterization and evaluation of the formulation
Dilution study
SEDDS formulations containing 10 mg of ATR (1 part)
were diluted with 10 parts of distilled water, 0.1 N HCl
and Phosphate buer of pH 6.8 and visually observed
(Table 2).[9]
Drug content
ATR from preweighed SEDDS formulations was extracted
by dissolving the formulations in 25 ml of methanol. ATR
content in the methanolic extract was analyzed spec-
trophotometrically (Shimadzu 1700, Japan) at 241 nm,
against the standard methanolic solution of ATR.[10,11]
Disintegration test
e test was performed according to procedure and spec-
ication mentioned in ocial compendia (USP 2005).
Self-emulsication and precipitation assessment
Assessment of the self-emulsifying properties of SEDDS
formulations were performed by visual assessment.[5,12]
Dierent formulations were categorized on the basis of
speed of emulsication, clarity, and apparent stability of
the resultant emulsion. Visual assessment was performed
by dropwise addition of the SEDDS into 250 mL of dis-
tilled water at room temperature, and the contents were
gently stirred magnetically at ∼ 100 rpm. Precipitation
was evaluated by visual assessment of the resultant emul-
sion after 24 h. e formulations were then categorized
as clear (transparent or transparent with bluish tinge),
non-clear (turbid), stable (no precipitation at the end of
24 h), or unstable (showing precipitation within 24 h).
Figure 2. Continued.
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Table 1. Composition of various SEDDS formulations.
Components (% W/W) F1 F2 F3 F4 F5 F6
ATR 1.9 1.9 1.9 1.9 1.9
1.9
Captex-355 39.3 49.1 44.11 53.9 37.2
50.9
Capmul-MCM 31.4 25.5 28.4 23.5 33.3
24.5
Tween-80 15.7 12.7 13.7 11.7 15.7
11.7
PEG-400 11.7 9.8 10.8 8.8 11.9
9.4
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Enhancement of oral bioavailability of atorvastatin calcium 69
Viscosity determination
SEDDS (1 mL) was diluted 10 and 100 times with the
distilled water in beaker with constant stirring on mag-
netic stirrer.[9] Viscosity of the resultant microemulsion
and undiluted SEDDS was measured using viscometer
(Brookeld-DV-E) (Table 3).
Droplet size analysis
Droplet size of SEDDS diluted with water was determined
using a photon correlation spectrometer (Zetasizer 3000
HAS, Malvern Ltd, UK) based on the laser light scattering
phenomenon.[1,3,10] Optimized formulation was diluted
200 times with puried water. Diluted samples were
directly placed into the module and measurements were
made in triplicate after 2-min stirring (Table 3).
Zeta-potential determination
SEDDS (1 mL) was diluted 10 times and 100 times with dis-
tilled water in beaker with constant stirring on a magnetic
stirrer.[9,13] Zeta-potential of the resulting microemulsion
was determined using the Zetasizer (3000 HAS, Malvern
Ltd, UK) (Figure 3).
In vitro dissolution studies
e purpose of in vitro dissolution study was to check the
dissolution rate of SEDDS.
e quantitative in vitro release test was performed
in 900 mL of phosphate buer pH 6.8 maintained at
37 ± 0.5°C using USP XXIV type II dissolution appara-
tus (Electrolab TDT-08L Plus, India). e paddles were
rotated at 100 rpm. e SEDDS formulations were lled
into transparent hard gelatin capsules (0 sizes) and used
for drug release studies; results were compared with
marketed ATR tablet (ZIVAST). Five mL aliquots were
collected periodically and replaced with fresh dissolution
medium. Aliquots, after ltration through Whatman lter
paper (No. 41), were analyzed spectrophotometrically at
241 nm for ATR content.[14]
In vitro drug diusion studies
e purpose of in vitro diusion study was to check the
permeation of drug through biological membrane. In
vitro diusion studies were carried out by using the dialy-
sis technique.[10] One end of pretreated cellulose dialysis
tubing (7 cm in length) was tied with thread and 0.5 mL
of self-emulsifying formulation (equivalent to 10 mg ATR)
was placed in it along with 0.5 mL of dialyzing medium
(phosphate buer pH 6.8). e other end of tubing was
also secured with thread and was allowed to rotate freely
in the dissolution vessel of a USP XXIV type II dissolu-
tion test apparatus (Electrolab TDT-08L Plus, India) that
contained 900 mL dialyzing medium (phosphate buer
pH 6.8) maintained at 37 ± 0.5°C and stirred at 100 rpm.
Placebo formulation (blank SEDDS, without drug) was
also tested simultaneously under identical conditions so
as to check interference, if any. Aliquots were collected
periodically and replaced with fresh dissolution medium
and analyzed spectrophotometrically at 241 nm for ATR
content.
In vivo studies
In vivo study was approved and performed in accord-
ance with the guideline of the animal ethics committee.
e study was conducted in three groups consisting of
six male albino rats weighing 150–200 g. Animals were
grouped as:
Group I: Six rats for pure ATR drug solution
(Reference);
Table 3. Characterization of SEDDS formulations.
Parameters F1 F2 F3 F4 F5 F6
Drug content (%) 92.8 ± 1.23 91.9 ± 2.83 92.5 ± 1.91 94.7 ± 2.15 96.98 ± 2.78 93.3 ± 2.43
Disintegration time (sec) 2.45 ± 1.72 2.33 ± 1.2 1.49 ± 0.9 3.5 ± 0.35 1.33 ± 1.1 2.13 ± 1.34
S.E.T. (sec) 49 ± 2 98±3 67±5 53±6 59±7 51 ± 1.8
Precipitation Unstable Stable Unstable Stable Stable Stable
Clarity Turbid Bluish Turbid Bluish Bluish Bluish
Viscosity (cps) 0 Times dilution 315 314 312 315 317 319
10 Times dilution 1.12 1.13 1.21 1.11 1.24 1.31
100 Times dilution 0.887 0.884 0.87 0.869 0.845 0.841
Droplet size (nm) 231.11 191.24 135.23 229.11 56.14 231.19
n = 3.
Table 2. Dilution study.
Vehicles F1 F2 F3 F4 F5 F6
Distilled water Stable up to 1 h Unclear within
30 min
Stable up to 3 h Unclear within
30 min
Stable up to 5 h
Unclear within
30 min
0.1 N HCl Stable up to 1 h Unclear within
30 min
Stable up to 3 h Unclear within
30 min
Stable up to 5 h
Unclear within
30 min
Phosphate buer
6.8
Stable up to 1 h Unclear within
30 min
Stable up to 3 h Unclear within
30 min
Stable up to 5 h
Unclear within
30 min
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70 P.J. Kadu et al
Group II: Six rats for self-microemulsifying drug
delivery system (SMEDDS) formulation of ATR (Test);
Group III: Six rats for placebo of SEDDS formulation
(Placebo);
Group IV: Six rats for Control (No formulation)
(Control)
e rats were fasted overnight and injected intraperi-
toneally 250 mg/kg Triton WR 1339 (Tyloxapol, Sigma
Chemical Co, St Louis, MO, USA) dissolved in 0.5% car-
boxy methyl cellulose (CMC) solution. Control groups
of rats were given the vehicle (CMC solution 0.5%), and
experimental groups were given pure ATR (25 mg/kg
body weight) or the SEDDS formulation (equivalent to
25 mg/kg ATR). e oral dosing was performed by intu-
bation using an 18-gauge feeding needle (the volume
to be fed was 1.0 mL in all cases). Blood samples were
drawn at 0, 24, and 48 h and serum was separated by
centrifugation at 10,000 g. Serum cholesterol, triglyc-
erides, low-density lipoprotein cholesterol (LDL- CH)
were estimated in all groups. Statistical analysis of the
collected data was performed using one way analysis of
variance (ANOVA).[5]
Stability studies
Chemical and physical stability of optimized ATR
SEDDS formulation was assessed at 40 ± 2°C/75 ± 5%
RH as per ICH Guidelines. SEDDS equivalent to 10 mg
ATR was lled in size ‘0’ hard gelatin capsules, packed in
aluminum strips and stored for three months in stabil-
ity chamber (CHM 10S, REMI Instruments Ltd, India).
Samples were analyzed at 0, 30, 60 and 90 days for drug
content, disintegration time and in vitro dissolution
prole.[15]
Results and discussion
Solubility studies
Solubility is the most important criteria for selecting the
vehicle for formulating a self emulsifying formulation.
erefore, the components used in the system should
have high solubilization capacity for the drug, ensuring
the solubilization of the drug in the resultant dispersion
(Figure 1). As seen from the results, Captex 355, Capmul
MCM, Tween 80 and PEG 400 showed the highest solu-
bilization capacity for ATR, as compared to other com-
ponents. us, for present study Captex 355 selected as
an oil, Capmul MCM and Tween 80 as surfactants and
PEG 400 used as co-surfactant.
Low HLB surfactants may also be an important com-
ponent of oral lipid-based
formulations by behaving as a coupling agent for the
high HLB surfactant and lipophilic solvent components
as well as contributing to solubilization by remaining
associated with the lipophilic solvent post dispersion.
Furthermore, using a blend of low and high HLB sur-
factants may also lead to more rapid dispersion and ner
emulsion droplet size on addition to an aqueous phase.
Pseudo ternary phase diagram
Self-microemulsifying systems form ne oil-water emul-
sions with only gentle agitation, upon their introduction
Results
Zeta Potential (mV):
Zeta Deviation (mV):
Conductivity (mS/cm):
Result quality: See result quality report
8.97
−22.1 −18.0
−30.8
0.000.0995
Mean (mV)
Peak 1:
Peak 2:
Peak 3:
63.4
36.6
0.0
Area (%) Width (mV)
6.75
4.02
0.00
Zeta Potential Distribution
100000
80000
60000
40000
20000
0
Total Counts
−200 −100 0 100 200
Zeta Potential (mV)
Record 2: SMEDDS 1
Figure 3. Zeta potential analysis of optimized formulation.
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Enhancement of oral bioavailability of atorvastatin calcium 71
into aqueous media. Surfactant and co-surfactant get
preferentially adsorbed at the interface, reducing the
interfacial energy as well as providing a mechanical barrier
to coalescence. e decrease in the free energy required
for the emulsion formation consequently improves the
thermodynamic stability of the microemulsion formula-
tion. erefore, the selection of oil and surfactant, and
the mixing ratio of oil to S/CoS, play an important role in
the formation of the microemulsion.[16] After performing
solubility studies, components in which drug showed
more solubility put forwarded for phase behavior study.
In the present study, combinations of surfactants (Smix)
with high and low HLB values were used. Capmul MCM
has low HLB value (5–6) and Tween 80 having higher
(15). e combination of low and high HLB surfactants
leads to more rapid dispersion and ner emulsion droplet
size on addition to aqueous phase.[17] Capmul MCM and
Tween 80 in the ratio of 2:1 showed wider microemul-
sion existence area and rapid emulsications compared
with1:1 and 3:1. us 2:1 Smix ratio along with cosur-
factant designated as S/Cos used in combination with
constant amount of Captex-355 in the ratio of 2:1, 3:1,
4:1 and 5:1 of S/Cos and Captex-355, respectively. Among
these 4:1 and 5:1 showed wider microemulsion existence
area. Figure 2 shows an increase in the microemulsion
area as increase in S/Cos ratio from 2:1 to 5:1. But in 5:1
ratio the concentration of surfactants goes beyond limit,
hence authors selected 4:1 S/Cos ratio for formulation.
PEG 400 is reported to be incompatible with hard gelatin
capsules when used in high concentrations (> 15% W/W
of total formulation).[5] us PEG 400 concentration was
kept below 15% w/w in formulation.
Dilution study
e objective of the dilution study was to study the degree
of emulsication and recrystallization of the drug, if any.
Viscosity of diluted and undiluted SMEDDS was meas-
ured to study the eect on emulsication time. Dilution
may better mimic conditions in the stomach following
oral administration of SMEDDS pre- concentrate.
Dilution study was carried out to access the eect of dilu-
tion on SMEDDS pre-concentrates. Accurate mixture of
emulsier is necessary to form stable microemulsion, for
the development of SEDDS formulation when one part
of each SEDDS formulation was diluted with 10 parts
of distilled water, 0.1 HCl and phosphate buer 6.8 pH
(Table 2). It implies that the formulation F5 was more
stable because there was no precipitation or cryalalliza-
tion of drug.[9]
Drug content
Drug content of the SEDDS formulations are shown in
Table 3, which was in the limit (98–102%).
Disintegration test
e disintegration time of SEDDS formulation is shown
in Table 3. e results demonstrate that formulation F5
showed less disintegration time (1.33 ± 1.1 sec) compared
with the other formulations.
Self-emulsication and precipitation assessment
It was found that the self-emulsication time (SET)
decreases with an increase in concentration of surfactants
up to 49%, beyond and below which there was turbid and
unstable dispersion. is may be due to excess penetra-
tion of water into the bulk oil causing massive interfacial
disruption and ejection of droplets into the bulk aqueous
phase. However, a higher level of surfactant decreases the
solubility limit of the drug and may subsequently lead
to precipitation. e decrease in self-emulsication time
can be assumed due to the relative increase in surfactants
concentration. Hence the combination of high and low
HLB value surfactants were used because low HLB sur-
factants were behaving as coupling agent for high HLB
surfactants. Furthermore, using a blend of low and high
HLB surfactants may also lead to more rapid dispersion
and ner emulsion droplet size on addition to an aque-
ous phase.[17]
As concentration of surfactant increases the molec-
ular volume increases which aects penetration at
the interface hence SET decreases. e ratio of Smix
of 2:1 and S/CoS of 4:1 was kept constant for initial
formulations.
Viscosity determination
Viscosity of SEDDS without dilution was found to be in
between 312 and 319 cP, which was suitable for lling in
hard gelatin capsule without risk of leaking problem. As
SEDDS was diluted 10 and 100 times with water, viscosity
of the system was decreased, which indicates that oral
administration of SEDDS formulation will be diluted
with the stomach uid and viscosity will be decreased
and therefore absorption from the stomach will be fast
(Table 3).
Droplet size analysis
e droplet size of the emulsion is a crucial factor in self-
emulsication performance because it determines the
rate and extent of drug release as well as drug absorption.
Also, it has been reported that the smaller particle size of
the emulsion droplets may lead to more rapid absorption
and improve the bioavailability.[13] It was observed that
increasing the S/CoS ratio led to decrease in mean drop-
let size. It is well known that in microemulsion systems
the addition of surfactants stabilize and condense the
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72 P.J. Kadu et al
interfacial lm, while the addition of cosurfactant causes
the lm to expand; thus, the relative proportion of sur-
factant to cosurfactant has varied eects on the droplet
size. e SEDDS was found to be clear transparent after
the 100 times dilution with distilled water and remained
stable (Table 3).
Zeta-potential determination
e magnitude of the zeta potential gives an indication
of the potential stability of the colloidal system. If all the
particles have a large negative or positive zeta potential
they will repel each other and there is dispersion stability.
If the particles have low zeta potential values then there
is no force to prevent the particles coming together and
there is dispersion instability. A dividing line between
stable and unstable aqueous dispersions is generally
taken at either +30 or −30mV. Particles with zeta poten-
tials more positive than +30mV are normally considered
stable. Particles with zeta potentials more negative than
−30mV are normally considered stable. Zeta potential of
the system was found to be −22.1 mV, which indicated
the droplets of microemulsion having negative charge,
which is closer to range.
In vitro dissolution studies
Drug release from the SEDDS formulation (F5) was
found to be signicantly higher (P) as compared with
that of Marketed ATR tablet (ZIVAST, FDC Ltd, India).
e result indicates that (F5) shows 70% of drug release
within 10 min and 97% of drug release in 60 min, which
is higher than the marketed formulation (39% and 61%
in 10 and 60 min, respectively). It could be suggested
that the SEDDS formulation (F5) resulted in spontane-
ous formation of a microemulsion with a small droplet
size, which permitted a faster rate of drug release into
the aqueous phase, much faster than that of Marketed
ATR tablet. e dramatic increase in the rate of release of
ATR from SMEDDS compared to marketed formulation
can be attributed to its quick dispersability and ability to
keep drug in solubilized state. us, this greater availabil-
ity of dissolved ATR from the SEDDS formulation could
lead to higher absorption and higher oral bioavailability
(Figure 4).
In vitro drug diusion studies
Conventional dissolution testing of SEDDS has a limita-
tion in mimicking its real time in vivo dissolution and
such a technique can only provide a measure of dispers-
ibility of SEDDS in the dissolution medium. Alternatively,
for evaluating the in vitro performance of SEDDS, drug
diusion studies using the dialysis technique are very
popular and well documented in many literatures.[10,16]
Diusion studies were performed for SEDDS F2, F4,
and F5, as these formulations show smaller droplet size
among other formulations. ough SEDDS F3 has less
droplet size than F2 it found unstable and turbid on
precipitation and clarity test, respectively. e release of
ATR from these dosage forms was evaluated in phosphate
buer pH 6.8; the release percentage of F5 was signi-
cantly higher than that of F2 and F4 (Figure 5). It could
suggest that ATR dissolved perfectly in SEDDS form could
be released due to the small droplet size, which permits a
faster rate of drug release into aqueous phase. e release
rate of ATR from SEDDS F5 (mean droplet size: 56.86 nm)
was faster than SEDDS F2 and F4 (mean droplet size: 191
and 231 nm, respectively). In this study, diusion proles
of all three formulations (F2, F4, and F5) did not show
any dierences during initial 2 h, however, at the end
of 12 h, formulation F5 showed about 97.07% diusion
against F2 and F4 shows 85.64% and 87.31% diusion,
respectively (Figure 5). Results clearly indicate the eect
of mean droplet size on drug diusion across dialyzing
membrane. Hence increasing the particle size of micro-
emulsion could decrease the release rate of drug and it
might suggest that release rate of drug could be control-
led by regulating the mean particle size.
120
100
80
60
40
20
0
% Cumulative Release
F1
F2
F3
F4
F5
F6
ZIVAST
010203040506070
Time (min)
Figure 4. In vitro dissolution studies of various developed and mar-
keted (ZIVAST) formulations.
120
100
80
60
40
20
0
% Diffusion
F2
F4
F5
02468101214
Time (h)
Figure 5. In vitro diusion studies of F2, F4 and F5.
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Enhancement of oral bioavailability of atorvastatin calcium 73
In vivo studies
e study was performed to evaluate the pharmacody-
namic potential of an optimized formulation (F5) against
pure ATR using a Triton-induced hyperlipidemia model.
Triton is a non-ionic surfactant that induces hyper-
lipidemia by inhibiting peripheral lipoprotein lipase
enzymes responsible for removal of lipid particles from
the body. e administration of Triton leads to transient
elevation of lipid levels. Hypolipidemic activity of ATR
causes reduction in elevated total CH, LDL-CH and TG
levels in blood. At the same time, it causes elevation
of plasma HDL-CH level, which promotes the removal
of cholesterol from peripheral cells and facilitates its
delivery back to the liver (Figure 6). As ATR has longer
half-life of 14 h, it is known to have a longer stay in
the blood circulation. us there is longer duration of
action due to an optimal initial plasma drug level. On
comparison with pure ATR, optimized SEDDS formu-
lation (F5) shows better results. Results indicates that
in 48 h ATR lowered cholesterol (78.61% inhibition),
triglyceride (63.77% inhibition), and LDL (49.65% inhi-
bition), and SEDDS formulation resulted in a greater
reduction of cholesterol (95.93% inhibition), triglycer-
ide (95.47% inhibition), and LDL (100.14% inhibition).
e higher lipid-lowering activity of the SEDDS is due
to complete dissolution of ATR in SEDDS, which could
have increased absorption and thereby led to a higher
plasma drug concentration (higher bioavailability).
e low bioavailability of ATR is attributed to its poor
aqueous solubility. e results of statistical analysis
of collected data using one way analysis of variance
(ANOVA) shows signicant dierence between pure
ATR and optimized SEDDS formulation (P < 0.001). e
above dierence in pharmacodynamic activity and the
results from in vitro dissolution studies suggest that the
SEDDS formulation shows better lipid lowering activity
than pure ATR.
Stability studies
Optimized formulation (F5) lled into hard gelatin cap-
sules as the nal dosage form. However liquid-lled hard
gelatin capsules are prone to leakage, and the entire sys-
tem has a very limited shelf life owing to its liquid char-
acteristics and the possibility of precipitation of the drug
from the system. us, the optimized formulation (F5)
was subjected to stability studies to evaluate its stability
and the integrity of the dosage form. No change in the
physical parameters such as homogeneity and clarity
was observed during the stability studies. ere was no
major change in the drug content, disintegration time,
and in vitro dissolution prole. It was also observed that
the formulation was compatible with the hard gelatin
capsule shells. Also, there was no phase separation, and
drug precipitation was found at the end of three-month
stability studies indicating that ATR remained chemically
stable in SEDDS (Table 4).
Conclusions
SEDDS appeared to be an interesting approach to
improve problems associated with oral delivery of ATR.
ATR SEDDS formulation was superior to marketed
formulation in respect to in vitro dissolution prole. It
also shows better in vivo hypolipidimic activity than
pure ATR. us, SEDDS can be regarded as a novel
and commercially feasible alternative to current ATR
formulations
Acknowledgements
e authors are thankful to R.C. Patel Institute of
Pharmaceutical Education and Research for providing
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
% Inhibition% Inhibition% Inhibition
24hrs 48hrs
Time(Hr)
Triglyceride
LDL Cholesterol
Cholesterol
ATR
F5
**
** **
**
**
**
**
**
**
**
**
**
Figure 6. In vivo study of ATR and optimized formulation F5.
Table 4. Stability studies.
Sampling points
Disintegration
time (sec) % Drug content % Drug release
0 day 1.33 ± 1.1 95.1 ± 1.83 96.98 ± 2.78
30 days 1.49 ± 0.9 94.7 ± 2.15 95.31 ± 3.35
60 days 2.33 ± 1.2 93.3 ± 2.43 93.7 ± 2.85
90 days 2.45 ± 1.7 91.9 ± 2.83 92.5 ± 1.91
n = 3.
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74 P.J. Kadu et al
facilities to carry out the research work. e authors are
also thankful to FDC Ltd (India), Abitec Corporation
(USA), Colorcon Asia Pvt. Ltd (Goa, India), and Associated
capsules (India) for providing gift samples of ATR, oils
and surfactants, Gelucire 44/14 and empty hard gelatin
capsules, respectively.
Declaration of interest
e authors report no conicts of interest. e authors
alone are responsible for the content and writing of the
pap e r.
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