Research Article

Formulation Development of Parenteral Phospholipid-based Microemulsion

of Etoposide
Jayesh Jain,
1
Clara Fernandes,
1
and Vandana Patravale
1,2
Received 25 December 2009; accepted 16 April 2010; published online 13 May 2010
Abstract. The aim of the present study was to investigate the potential of a phospholipid-based
microemulsion formulation for parenteral delivery of anticancer drug, etoposide. The microemulsion
area was identied by constructing pseudoternary phase diagrams. The prepared microemulsions were
subjected to different thermodynamic stability tests. The microemulsion formulations that passed
thermodynamic stability tests were characterized for optical birefringence, droplet size, viscosity
measurement, and pH measurements. To assess the safety of the formulations for parenteral delivery,
the formulation was subjected to compatibility studies with various intravenous infusions and in vitro
erythrocyte toxicity study. The developed formulation was found to be robust and safe for parenteral
delivery.
KEY WORDS: etoposide; microemulsion; parenteral; phospholipid.
INTRODUCTION
Etoposide (Fig. 1), an epipodophyllotoxin, is an anti-
cancer drug useful for the treatment of small cell lung cancer
and testicular carcinoma. Prior to administration, the drug has
to be diluted in the infusion uid; its low aqueous solubility
thus acts as a constraint in the formulation of its parenteral
dosage form. This attribute results in drug precipitation in the
infusion uid thereby proving detrimental to the health owing
to the possibility of capillary blockade. The currently
marketed formulation contains 20 mg/ml etoposide in non-
aqueous vehicle including 2 mg citric acid, 30 mg benzyl
alcohol, 80 mg polysorbate 80, 650 mg polyethylene glycol
300, and 30.5% (v/v) alcohol (1). The components of this
formulation have been associated with serious side effects
such as pain, inammation, tissue damage, necrosis at the site
of injection, and substantial hemolysis (2). Furthermore, the
risk of precipitation as microcrystals encountered on the
dilution of the organic solvents phase with consequent venous
sequelae and too rapid an infusion of etoposide precipitates
hypotension of the patient (3). Hence, an etoposide prepara-
tion compatible with the infusion uids would serve as an
attractive alternative to the marketed system (Fig. 1).
Recently, much attention has been paid to the application
of microemulsions as drug delivery systems, since microemul-
sions are thermodynamically stable and are formed sponta-
neously by simple mixing of the various components (49).
Thus, the focus of the present investigation was to improve
the solubility of etoposide, a poorly water-soluble drug without
chemical modication and by using microemulsion drug
delivery system that is suitable for parenteral administration
and stable after dilution with commonly used infusion uids.
MATERIALS AND METHODS
Materials
Etoposide was a gift from Cadila Pharmaceuticals, India,
Epikuron135 F (uid phosphatidyl choline enriched leci-
thin) was a kind gift from Degussa, Capmul MCM was a
kind gift from Abitec Corp. U.S.A., Tween 80 and Poly-
ethylene Glycol 400 (PEG 400) were purchased from s.d.
Fine chemicals. All other chemicals were of AR-grade and
purchased from Merck.
Solubility Studies
The xed amount of surfactant/oil was weighed accu-
rately and transferred to a small test tube. In this test tube,
the drug was added to assess the equilibrium solubility of the
drug at ambient condition of 25 to 27C. Thereafter, the
amount of surfactant/oil required to solubilize the drug was
determined. Table I depicts the solubility data compounded
for etoposide in various oils, surfactants, and solubilizers.
Preparation of Microemulsion
Formulation Methodology
Etoposide was solubilized in PEG 400 by vortexing.
Thereafter, oil was mixed, followed by surfactant and
1
Department of Pharmaceutical Sciences and Technology, Institute of
Chemical Technology (Autonomous), Matunga, Mumbai 400 019,
India.
2
To whomcorrespondence should be addressed. (e-mail: vbp_muict@yahoo.
co.in)
AAPS PharmSciTech, Vol. 11, No. 2, June 2010 (
#
2010)
DOI: 10.1208/s12249-010-9440-x
1530-9932/10/0200-0826/0 # 2010 American Association of Pharmaceutical Scientists 826
phospholipid. The resultant solution was mixed uniformly by
vortexing. Then, the xed amount of water was added and
gently mixed to obtain a microemulsion. The resultant
microemulsion was sterilized by autoclaving at 121C and
15 psi for 15 min.
Preparation of Pseudoternary Phase Diagram
The pseudo-ternary phase diagrams of oil, surfactant:
cosurfactant [PEG 400: Tween80: Epikuron135F (16:9:1)],
and water were constructed prior to optimization of the
formulation. Surfactant was mixed with cosurfactant in xed
weight ratios. Aliquots of each surfactantcosurfactant mix-
ture were then mixed with oil and nally with aqueous phase.
Mixtures were gently shaken or mixed by vortexing and kept
at ambient temperature (25C) to attain equilibrium. The
equilibrated samples were assessed visually for their appear-
ance, i.e., it was noted as being clear and transparent
microemulsions, or crude emulsions or gels.
Characterization of Microemulsion
Optical Birefringence
The microemulsion was placed between two polarizing
plates and then observed for light transmittance. After this,
one of the plates was rotated relative to the other through 90
(crossed polarizer) and then examined.
Determination of Particle Size
Photon correlation spectroscopy using laser light scatter-
ing is frequently used to determine particle size of emulsions.
A Beckman N4 Plus submicron Particle Size Analyzer was
employed to monitor particle size of the microemulsion. The
instrument calculated the mean particle size and polydisper-
sity from intensity, assuming spherical particles. Polydisper-
sity is a measure of particle homogeneity and it varies from
0.0 to 1.0. The closer to zero the polydispersity value, the
more homogeneous are the particles. Prior to analysis,
samples were diluted vefold using 0.22 ltered double
distilled water. Light scattering was monitored at 90 scatter-
ing angle and temperature of 25C or 37C. All measure-
ments were performed in triplicate.
Accelerated Stability Testing
Centrifugation
Centrifugal methods have been employed to subject the
system to assess accelerated stability. The microemulsion was
subjected to centrifugation at 5,175g for 30 min and
observed for any separation, i.e., incompatibility (8).
FreezeThaw Cycling
A 5-ml microemulsion was subjected for six heating/
cooling cycles between 40C and 4C with storage at each
temperature for not less than 24 h and assessed for their
physical instability like phase separation and precipitation (8).
Viscosity Measurements
The viscosity of the formulation was ascertained by an
Oswald-type viscometer (Techniko 841, D). The viscometer
tube was lled with the exact amount of the formulation.
The meniscus of the liquid in capillary tube was adjusted
to the level of the top graduation mark with the aid of
vacuum. The time in seconds was recorded for liquid to ow
from the upper mark to the lower mark in the capillary tube.
The time required for water was also recorded. The
procedure was repeated three times and the average was
taken for calculation.
pH Measurement
The acceptable range is pH 212 for intravenous and
intramuscular administration whereas for subcutaneous
administration, the range is reduced to pH 2.79.0 as the rate
of in vivo dilution is signicantly reduced resulting in more
potential for irritation at the injection site.
The pH of formulation was measured using by Systronic
Digital pH meter 335, standardized using pH 4.0 and 7.0
standard buffers use.
Compatibility Assessment with Different Injectable Diluents
The dilutability and compatibility of developed formula-
tions with the 0.9% sodium chloride injection or 5% dextrose
Table I. Solubility Data of Etoposide in Oils/Surfactants/Solubilizers
Oils/surfactants/solubilizers Solubility (mg/mL)
Miglyol-812 4
Soyabean oil 1.5
Capmul MCM 10
Epikuron 135 F 5.5
PEG-400 110
Tween-80 35
Tween-20 30
Cremophor EL 23
Fig. 1. Chemical structure of etoposide
827 Formulation Development of Parenteral Phospholipid-based Microemulsion of Etoposide
injection was assessed by diluting the developed microemulsion
formulation in different concentration ranges (0.21 mg/ml)
with 0.9% sodium chloride injection and 5% dextrose injection
and kept them for visual inspection with respect to phase
separation or precipitation for a period of 3 h.
In Vitro Erythrocyte Toxicity Study
The erythrocyte toxicity assay was conducted as
described by Bock et al. (10). Fresh blood was collected in
the vial containing EDTA (anticoagulant). Red blood cells
(RBCs) were isolated by centrifugation (5,000 rpm for 5 min)
and the RBCs were washed three times with isotonic
phosphate buffer pH 7.4 before diluting with buffer to
prepare erythrocyte stock dispersion (three parts of centri-
fuged erythrocytes plus 11 parts buffer). The buffer consisted
of Na
2
PO
4
10H
2
O (7.95 g), KH
2
PO
4
(0.76 g), NaCl (7.2 g),
and distilled water (add 1,000 ml). The washing step was
repeated in order to remove debris and serum protein. A 100-
l aliquot stock dispersion was added per milliliter of test
sample. The resulting solution was incubated at 37C for a
period of 1 h. After incubation under shaking, debris and
intact erythrocytes were removed by centrifugation. One
hundred milliliters of resulting supernatant was added to
2 ml of an ethanol/HCl mixture [(39 parts ethanol (99% v/v)+
1 part of HCl (37% w/v)]. This mixture dissolved all
components and avoided the precipitation of hemoglobin.
The absorbance of the mixture was determined at 398 nm by
spectrometer monitoring against a blank sample. Control
sample of 100% lysis (in TritonX 100) was employed in the
experiment. The percentage of hemolysis caused by the test
sample was calculated by following equation:
Hemolysis caused by sample %
Abs of the test sample=Abs at 100% lysis 100
Test for Sterility
Test for sterility was performed by using nutrient broth
medium and incubated at 37C for 7 days. For the study,
groups including developed microemulsion, negative control
medium, and positive control medium incubated with Bacillus
subtilis were evaluated.
RESULTS AND DISCUSSION
Solubility Studies
Parenteral administration of microemulsion requires the
component such oil, surfactant(s) and co surfactant(s) to be
biocompatible and safe. In this regards, nonionic or zwitter-
ionic surfactants have found to favorable for pharmaceutical
applications since they are less toxic and less affected by
changes in pH and ionic strength (2).
Moreover, for a lipophilic drug, the principle objective is
to achieve a formulation where the drug is dissolved in the
liquid vehicle. By selecting the optimum liquid vehicle
composition, it is possible to minimize or eliminate precip-
itation of the drug. As shown in Table I, among the limited
choice of excipients, Capmul MCM was selected as the oil
component. However, as the dose of the drug was 20 mg, the
oil component was insufcient to solubilize the drug, hence
Tween80 and PEG 400 was selected as surfactant and
solubilizer components, respectively. In the process of for-
mulation, it was observed that drug precipitated in most of
the microemulsion formed by varying the combinations of
aforementioned excipients. In order to form a stable micro-
emulsion, complex interfacial lm of mixture of surfactants is
favorable and therefore an amphiphilic surfactant, Epi-
kuron135 F (a transparent, liquid fraction of soybean
lecithin and soybean oil with enriched phosphatidylcholine
content), having miscibility with oil, was chosen.
Preparation of Pseudoternary Phase Diagram
Phase studies were done to investigate the effect of
surfactant to cosurfactant ratio on the extent of stable o/w
microemulsion region. The microemulsions in the present
study formed spontaneously at ambient temperature when
their components were brought into contact. The areas of
microemulsion and isotropic regions increased with increasing
ratio of surfactant: cosurfactant (Fig. 2). It indicates that the
maximum proportions of oil incorporated in microemulsions
increased signicantly with increasing ratio of surfactant and
cosurfactant to oil. It is recommended that etoposide be
administered by slow intravenous infusion over a period of 30
to 60 min to prevent hypotension. Hence, using a constructed
phase diagram, the optimum ratios of the components for
microemulsion which would remain stable and prevent drug
precipitation over innite dilution was selected (Table II).
Fig. 2. Pseudoternary phase diagram
Table II. Composition of Parenteral Microemulsion of Etoposide
Component mg/mL % w/v
Drug 20 2
PEG-400 400 40
Capmul MCM 50 5
Epikuron 135 F 25 2.5
Tween-80 225 22.5
Water for injection q. s. to 1 ml 33
828 Jain, Fernandes and Patravale
Characterization of Microemulsion
Optical Birefringence
Birefringence (6) is a light-scattering phenomenon. It is
also called as double refraction and is found in liquid crystals
and anisotropic systems. In birefringence, the light passing
through a material is divided into two components with different
velocities and hence different refractive indices. Thus, when a
liquid crystal is observed between crossed polarizer, intense
bands of colors are seen which is known as birefringence. In
contrast, microemulsion appears completely black.
The developed microemulsion appeared completely dark
when observed between cross-polarizing plates validating that
the formulation was an isotropic, colloidal dispersion.
Determination of Particle Size
It is known that the particle size is distribution one of the
most important characteristics emulsion for the evaluation of
its stability and also in vivo fate of emulsion. Furthermore, it
is also well documented that size plays a pivotal role in the
circulation time of the particulate carriers. Some literature
states, particulate carriers with smaller size can evade
recognition by MPS; consequently have longer circulation
time in the bloodstream. In addition, smaller size eludes
capillary blockade resulting in attenuation of adverse effects
often associated with intravenous administration of partic-
ulate carriers. Therefore, the particle size of microemulsion
was assessed in triplicates. Table III depicts the particle size
before and after autoclaving. There was marginal increase in
particle size of the microemulsion globule; however, it was
lower than 100 nm.
Accelerated Stability Testing
Centrifugation
Centrifugal methods (8) have long been employed by
emulsion technologists to induce and accelerate instability by
gravitational means. It is commonly accepted that shelf life
under normal storage conditions can be predicted rapidly by
observing the separation of disperse phase when the micro-
emulsion is subjected to centrifugation.
The technique to determine behavior of small particles in
the gravitational force, i.e., their separation rates, is quite
simple and inexpensive providing a rapid fool-proof identi-
cation of the systems as microemulsions. Brownian move-
ment is associated with particles smaller than 0.5 m. The
microparticles in this size range are small enough to absorb
kinetic energy from bombardment by the molecules of
dispersion medium. It has been calculated that it causes such
a particle to change direction 10
24
times per second. This
keeps the dispersed droplets in a state of violent motion
preventing their settling under gravitational eld. So long as
they do not coalesce, it is Brownian movement that keeps the
droplets of microemulsion droplets from settling or creaming.
The reason that microemulsion droplets do not coalesce is
due to surface free energy of a microemulsion system. Just as
soon as two droplets coalesce to form a single droplet of
larger size, the interfacial tension of the new droplet becomes
negative, i.e., the system has negative free-surface energy.
The larger droplet now spontaneously increases its curvature
to effect zero interfacial tension again and two droplets of the
original size result. This process appears continuously as does
the bombardment of droplets by molecules of dispersion
medium. It is this dynamic equilibrium that keeps the
microemulsion systems stable.
At the end of 30 min, the developed microemulsion
showed absence of phase separation and drug precipitation
after centrifugation at 3,000 rpm, verifying the stability of the
formulation.
FreezeThaw Cycling
This test induces stress in the microemulsion (8). At
temperature below freezing, the formation of ice crystals in an
o/w type microemulsion may cause oil particles to elongate and
atten. In addition, the lipophilic portion of the emulsier
molecule will lose their mobility while the hydrophilic portions
are simultaneously dehydrated due to the freezing action of
water. As the sample is thawed, water is released and travels
rapidly through the microemulsion. If the system can heal
itself before coalescence occurs, then the microemulsion sur-
vives the test. However, if the rate of redissolution of the
ingredients is slow, instability may occur in case of micro-
emulsion which is not related to normal temperature processes.
The developed formulation did not show any evidence of
instability, the physical integrity of the formulation was
maintained at the end of the cycle.
Table III. Globule Size and Polydispersity Index of Developed
Formulation
Samples
Particle size
(nm)
Polydispersity
index (PI)
Formulation before autoclaving 58.2 0.912
Formulation after autoclaving 68.0 0.978
Table IV. Drug Content Stability Data
Conditions
% Drug content
Initial 7 days 1 month 2 months 3 months
RT 102.050.66 99.930.32 97.600.73 99.310.84 97.200.91
30C/60%RH 99.040.41 98.700.33 99.370.96 100.881.34
40C/75%RH 103.740.29 101.340.51 98.320.88 102.860.73
RT room temperature, RH relative humidity
829 Formulation Development of Parenteral Phospholipid-based Microemulsion of Etoposide
Viscosity Measurements
The stability of the microemulsion is often governed by
its viscosity, i.e., is an expression of the resistance to ow. The
viscosity denes the tendency of the system to agglomerate.
Moreover, for the developed microemulsion, the viscosity
measurement was of utmost importance, since it had to be
diluted using infusion uids prior to administration. It is well
known that the viscosity of parenteral formulation may also
affect the syringeability. Using the following equation, the
viscosity of the developed formulated was calculated to be
106.92 cP. The viscosity was calculated from the equation:

1t1
2t2
where
1
and
2
are the viscosities of the test and the
standard liquids,
1
and
2
are the densities of the liquids, and
t
1
and t
2
are the respective ow times in seconds.
The low viscosity of the developed microemulsion
ensures ease of syringeability as well as ease of mixing with
intravenous uids with minimum mechanical agitation.
pH Measurement
The pH of formulation measured regularly over a period
of 10 days showed that the pH of the formulation was in an
acceptable range for intravenous administration. For etopo-
side, pH plays a pivotal role in the drug stability. The pH for
stability of etoposide was found to be pH 5.4 which is
considered optimum to prevent the degradation of drug
(11). Due to constant pH, the drug content (Table IV) was
found to be in acceptable limits over a period of 3 months.
Compatibility Assessment with Different Injectable Diluents
As prescribed, etoposide for injection needs to be diluted
for administration by intravenous infusion in either 5%
dextrose injection or 0.9% sodium chloride injection to
produce a solution containing 200 to 400 g (0.2 to 0.4 mg)
of etoposide per milliliter. This is essential to prevent
hypotension, a side effect of the drug. As shown in Tables V
and VI, the diluted solution was stable for sufcient time
enabling slow intravenous infusion of etoposide in concentra-
tion range up to 1 mg/ml for 1.5 and 2 h in 0.9% sodiumchloride
injection and 5% dextrose injection, respectively. However, it
should be noted that at the recommended concentration of
0.4 mg/ ml, there was absence of drug precipitation for 3 h in
0.9% sodium chloride injection and 5% dextrose injection,
respectively. This clearly reects the superiority of the devel-
oped formulation over the existing etoposide injection which
reports drug precipitation as its limitations.
In Vitro Erythrocyte Toxicity Study
Colloidal drug carrier systems serve to minimize the side
effects of drugs used for parenteral applications. Side effects
often result from the destruction of corpuscles of blood or
tissue cells at the site of injection and these may be reduced
by incorporating the drug in the colloidal carriers (e.g.,
microemulsions). To corroborate this statement, the hemo-
lytic activity was done for estimating the membrane damage
caused by formulation in vivo. Since phospholipid and PEG-
400 are known for their relative non-toxic nature, they were
not considered for this study (2). However, the samples tested
for erythrocyte toxicity; developed microemulsion, Capmul
MCM andTween-80 showed considerably less hemolytic
activity (Table VII). This study indicated the safety of the
developed microemulsion for parenteral administration.
Test for Sterility
Sterility is one of the prerequisites for the parenteral
preparations. However, at extreme temperatures, phase
separation may occur but the microemulsions spontaneously
return to their initial state when brought back to normal
temperature and on adequate mixing. As it is established,
microemulsions can be sterilized by autoclaving (12); the
developed microemulsion was sterilized by autoclaving at
121C and 15 psi for 15 min. Although there was phase
separation after autoclaving, after shaking it gave a homoge-
neous microemulsion. The sterility testing of this sterile
microemulsion showed absence of microbial growth indicat-
ing the effectiveness of autoclaving. Furthermore, this was
attested by the stability of the developed microemulsion over
a period of 3 months (Table IV).
Table V. Compatibility of Microemulsion with 0.9% Sodium
Chloride Injection
Conc. of drug mg/mL
Time in hours
0.5 1 1.5 2 2.5 3
0.2 C C C C C C
0.4 C C C C C C
0.6 C C C C C C
0.8 C C C C C P
1 C C C P
C clear, P precipitation
Table VI. Compatibility of Microemulsion with 5% Dextrose
Injection
Conc. of drug mg/mL
Time in hours
0.5 1 1.5 2 2.5 3
0.2 C C C C C C
0.4 C C C C C C
0.6 C C C C C C
0.8 C C C C C P
1 C C C C P P
C clear, P precipitation
Table VII. Comparative Hemolysis after 1 Hour Incubation Period
Component % Hemolysis after 1 hour of incubation
Capmul MCM 0.16
Tween 80 0.05
Microemulsion 0.15
TritonX 100 6.69
830 Jain, Fernandes and Patravale
CONCLUSION
The parenteral phospholipid-based microemulsion was
successfully developed with particle size less than 100 nm.
The developed formulation was amenable to sterilization by
autoclaving and was found to be robust to dilution with
intravenous uids. The in vitro erythrocyte toxicity study
demonstrated the safety and acceptability of the formulation
for parenteral administration.
ACKNOWLEDGMENTS
The authors are grateful to the University Grant Com-
mission (New Delhi, India) for providing nancial support for
this project. The authors are also thankful to Degussa and
Abitec Corp. USA for providing the gift samples of oils,
surfactants, and cosurfactants.
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831 Formulation Development of Parenteral Phospholipid-based Microemulsion of Etoposide