Preparation and characterization of a triamcinolone acetonide palmitate submicron emulsion
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Triamcinolone acetonide submicron emulsion/Asian Journal of Pharmaceutical Sciences 2010, 5 (2): 61-73
Preparation and characterization of a triamcinolone
acetonide palmitate submicron emulsion
Cuilian Peng, Xiaonan Yan, Xing Tang*
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
Received 9 October 2009; Revised 18 January 2010; Accepted 14 March 2010
_____________________________________________________________________________________________________________
Abstract
Purpose: This investigation focused on the formulation and characterization of a triamcinolone acetonide palmitate (TAP) emulsion as
well as its long-term stability. Methods: In this study, the TAP emulsion (TAPE) was prepared using a high-pressure homogenizer and
the effects of operational parameters, such as homogenization pressure, number of cycles and sterilization methods, on the characteristics
of the TAPE involving the mean particle size, 99% diameter and particle size distribution (PSD) width as well as the generation of
lysophosphatidylcholine (LPC), were investigated. Results: The formulation of the TAPE consisted of TAP 0.4% (w/v, based on TAA),
MCT 15.0% (w/v), LCT 5.0% (w/v), soybean lecithin 3.0% (w/v), Pluronic F68 (F68) 0.4% (w/v) and glycerol 2.5% (w/v). The TAPE
was prepared using a two-stage high-pressure homogenizer with a homogenization pressure of 800 bar for 8 cycles, followed by steam
sterilization in a 121˚C rotating water autoclave for 10 min. This produced a stable TAPE with a mean particle size of 162 ± 38.22 nm
and a zeta-potential of –35.56 mV, and the stability testing at 4 ± 1˚C for twelve months proved that the TAPE remained stable and was
still extremely finely dispersed for parenteral administration. Conclusion: All these results indicated that the submicron emulsion is a
successful parenteral drug delivery system for TAP.
Keywords: Triamcinolone acetonide palmitate; Submicron emulsion; Formulation; Characterization; Stability
_____________________________________________________________________________________________________________
1. Introduction of the dosing regimens reflect the sustained and delayed
release function of TAA suspensions due to the systemic
Triamcinolone acetonide (TAA, Fig. 1A), a very accumulation of TAA, which is closely related to the
potent intermediate-acting glucocorticoid, is used systemic side effects induced by the suppression of the
topically or systemically for its anti-inflammatory hypothalamus-pituitary-adrenal (HPA) axis for at least
effects in disorders of many organ systems and skin [1]. 30 d [1]. This formulation is practically insoluble in
It can quickly relieve symptoms by blocking the water and, hence, provides a depot effect with constant
production of a variety of substances in the body that release of TAA from the injection site over a long period
cause inflammatory disorders [2]. The depot intra- of time, and its most common and most intensively
muscular (IM) formulation of TAA, e.g. Kenolog-10 studied uses are intra-articular (IA) administration for
(triamcinolone acetonide injectable suspension, USP30), the treatment of joint disorders and intravitreal injection
is available as a sterile crystalline suspension with a for several exsudative diseases of the eye [1]. However,
particle size of 5–10 μm [3]. The IM administration for it has failed to treat some acute or severe inflammations (i.e.
asthma involves a dose of 40 mg to 80 mg TAA as a acute asthma) that require a rapid pharmacodynamic
suspension to be given once a week [4]. The frequencies effect [5, 6]. So, more interest has recently focused on a
TAA prodrug, such as phosphate as well as palmitate,
__________ for intravenous (IV) administration. TAA-21-dihydrogen
*Corresponding author. Address: School of Pharmacy, Shenyang phosphate (TAA-DHP) is a water-soluble prodrug and
Pharmaceutical University, No. 103, Wenhua Road, Shenyang
it has been reported to be hydrolysed in less than 5 min
110016, China.
Tel: + 86-24-23986343; Fax: +86-24-23911736 in vivo [1]. Moreover, this intravenous administration
E-mail: tangpharm@yahoo.com.cn can completely change the metabolism of the active
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agent for the half-life of TAA-DHP after IV adminis- and middle-chain triglycerides (MCT), and the phospho-
tration (14 h) was shorter than that of IM TAA (125 h), lipids are used as emulsifiers to reduce the interfacial
resulting in a slight suppression of the HPA axis (48 h) [1]. tension, leading to significant enhancement in parenteral
Triamcinolone acetatonide palmitate (TAP, Fig. 1B), a nutrition [13]. Furthermore, emulsions prepared by
lipid-soluble C-21 palmitate prodrug of TAA, has been homogenization are preferable for industrial-scale
reported to be incorporated into liposomes with the methods because it is a cost-effective and technically
encapsulation efficiency increased from 5% (free simple approach and it offers a more physically stable
TAA) to 85% (TAP) for intra-articular treatment of an and safer product than solvent mixture products [14].
experimentally-induced arthritis in the knee joints of A series of lipid emulsions has been developed and
rabbits. In addition, this liposomal formulation was applied in clinical situations involving diazepam,
demonstrated to be more efficient than free TAA in etomidate and propofol because of their unique
solution in suppressing arthritis and was retained in properties, including low toxicity, reduced irritation and
the articular cavity for a much longer period because improved patient compliance [15-17]. So, this paper
of the long lipophilic chain of palmitate [7]. However, describes the development of a new formulation of
comparison of the drug loading efficiency of TAP TAP emulsions, offering a relatively high drug loading
in liposomes (0.03%, w/v) with the therapeutic dose efficiency and the ability to withstand thermal steam
indicates (2.5 mg to 5 mg for arthritis) retarded release sterilization required for IV and IA administration.
of TAP liposomes as well as their potential of clinical The present study involved an investigation of a TAP
efficiency [8]. emulsion including its formulation and characterization
Much attention has been given recently to the use of as well as its stability. The TAPE was prepared using
lipid submicron emulsions in drug delivery because of a high-pressure homogenizer and it survived thermal
their ability to incorporate drugs with poor solubility steam sterilization at 121˚C for 10 min and remained
within the oil phase, which could provide a high drug- stable in terms of both its physical and chemical
loading efficiency without the need for potentially toxic properties.
excipients [9]. Simultaneously, lipid emulsions can also
enhance the solubilization or stabilization of the incor- 2. Materials and methods
porated drugs to obtain sustained-release and targeting
effects [10]. Moreover, by using lipid emulsions, direct 2.1. Materials
contact of the drug with the body fluids and tissues
can also be avoided thereby minimizing possible side TAP was a kind gift from the China Pharmaceutical
effects [11, 12]. In addition, the oil phase of the emulsion University (Nanjing, China). Soybean lecithin
is mainly composed of long-chain triglycerides (LCT) (Epikuron 170, PC72%) was purchased from Degussa
O O
C H2
HO Me 14 O Me
Me
Me O Me
O O
HO Me HO Me
O O
Me Me
F F
O O
(A) (B)
Fig. 1. Structure of triamcinolone acetonide (A) and triamcinolone acetonide palmitate (B).
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Food Ingredients (Shanghai, China). Pure egg was transferred to vials after adding nitrogen gas and
lecithin of PL-100 M (PC 78%) was purchased from sterilized in a 121˚Crotating water steam autoclave for
Shanghai Advanced Vehicle Technology Ltd. Co 10 min.
(Q.P. Corporation, Japan). LCT was obtained from
Tieling Beiya Foods Ltd. (Liaoning, China), MCT 2.3. Lecithin types and sterilization stability
and Lipoid E80 (PC more than 80%) were purchased
from Shanghai Dongshang Shiye Company (Lipoid Egg yolk lecithin lipoid E80 (E80) and PL-100M as
Co., Germany), Poloxamer 188 (Pluronic F68) (BASF well as soybean lecithin Epikuron 170 were employed
AG, Ludwigshafen, Germany), Tween 80 (Shenyu to investigate the influence of different types of
Medicine and Chemical Industry Ltd. Co., Shanghai, phospholipids on the physical and chemical stability.
China), glycerol (Zhejiang Suichang Glycerol Plant, Three test TAPEs were prepared as described in Section
Zhejiang, China), and sodium oleate (Nation Drug 2.2. Then, the samples were separately sterilized by
Group Chemical Agents Ltd., Co., Shanghai,China) different methods including autoclaving in a 121˚
were obtained from the sources indicated while C rotating steam autoclave for 10 min and 20 min,
lysophosphatidylcholine (LPC) was obtained from in a 117˚C rotating steam autoclave for 30 min and
Sigma Co-Aldrich Trading Co. Ltd. Beijing, China). in a 100˚C rotating water bath for 45 min. The three
All other chemicals and reagents were of analytical or formulations above were all combined with 0.4% (w/v)
chromatographic grade. F68 as a co-emulsifier, 0.05% sodium as a stabilizer and 2.5%
(w/v) glycerol for adjustment of the osmotic pressure.
2.2. Preparation of a TAP submicron emulsion Then, all the samples were analyzed by HPLC before
and after sterilization.
The TAPE used in this paper was prepared using a
high-pressure homogenizer [18]. The aqueous phase (80%, 2.4. Characterization of the TAPE
w/v) and oil phase (20%, w/v) were separately prepared.
The aqueous phase consisted of double distilled water, 2.4.1. Particle size analysis and zeta potential
and a co-emulsifier and osmolarity adjustment agent
(glycerol), while the oil phase consisted of LCT and The particle size distribution (PSD) of the TAPE
MCT in a ratio of 5:15 (20 % of the total emulsion), was measured by the PCS technique using a NicompTM
lecithin (3%, w/v), and TAP (0.4%, w/v based on TAA). 380 Particle sizing system (Zeta Potential/Particle
The two phases were heated separately to 75˚C and Sizer NicompTM 380ZLS, Santa Barbara, California,
then the coarse emulsion was prepared by high shear USA). The system covered the range from 5 nm to
mixing (ULTRA TURRAX® T18 basic, IKA® WORKS approximately 3 um. The emulsion sample was diluted
Guangzhou, Germany) by rapidly adding the water 1:5000 with double-distilled water immediately before
phase to the oil phase at 10 000 r/min. The high shear the measurement at 25˚C. The effects of the pressure
mixing process was carried out for 3 min and repeated and the number of passes through the high-pressure
twice. The final emulsion was obtained in a high- homogenizer on the particle size of the droplets were
pressure homogenizer using Niro Soavi NS 10012K investigated. The NicompTM 380 system was also used
homogenization (Niro Soavi S.P.A., Via M.da Erba to determine the zeta potential (ζ-potential) by the ELS
Edoari, 29/A 43100 PARMA, Italy) at 800 bar for eight technique. Before measurement, the double-distilled
cycles. The temperature of the entire homogenization water used to dilute the emulsion samples was adjusted
process was kept at 40˚C using an ice-water bath. Then, to the same pH value as the emulsion using 0.1 M HCl
the volume was adjusted to 100 ml with double-distilled or NaOH solutions. Then, the emulsion sample was
water and the pH of the final emulsion was adjusted to 8.0 diluted 1:50 with the water as described above. The
with 0.1 M HCl or 0.1 M NaOH. Finally, the emulsion determination was carried out at 25˚C. The pH of the
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bulk emulsion was measured using a pH-meter (Leici®, 2.5. HPLC method
Shanghai Precision Science Instrument Ltd., Shanghai,
China) fitted with a microelectrode at room temperature 2.5.1. HPLC method for determination of the TAP
(25 ± 1˚C). concentration and EE
2.4.2. Drug concentration and encapsulation efficiency An HPLC system (Hitachi D-7000) with a Kromasil
(EE) C18 column (5 mm, 4.6 mm × 250 mm) (Dalian, China)
consisted of an autosampler (L-7200), a pump (L-7100),
The EE of the TAPE was obtained by measuring the and a UV detector (L-7420), all interfaced with D-7000
free TAP concentration in the dispersed medium. The HSM software. The mobile phase was 100% methanol.
emulsions with drugs were centrifuged at 50 000 r/min The flow rate was 1.0 ml/min and the UV detector was
and 4˚C for 90 min in a CS120GXL ultracentrifuge (Hitachi set at 254 nm, while the column temperature was 25 ± 1
Co. Japan) in order to separate the incorporated drug ˚C and the injection volume was 10 μl.
from the non-incorporated drug [19]. The subnatant
(water phase) was analyzed without dilution by HPLC 2.5.2. HPLC method for the TAPE lysophospholipid
to obtain the free TAP concentration, and the EE was
calculated from Equ. 1 [20]. The total content of TAP An HPLC (Jasco LC-Net II/APC) system using a
in the TAPE was determined by diluting the TAPE Kromasil C18 column (5 mm, 4.6 mm × 250 mm) (Dalian,
100-fold with methanol and carrying out an analysis by China) consisted of an autosampler (Jasco 2055 plus),
HPLC. a pump (Jasco 2089 plus) and an ELSD detector (2000
ES, Jasco) and this was used to determine the LPC
(Eq.1) C totalV total C waterV water h100 (Eq.1)
content of the TAPE. The initial mobile phase gradient
EE% =
C totalV total ratio was 82:18 for solvents A and B respectively, where
Where Ctotal and Cwater represent the concentration A is methanol and B is distilled water. The elution
of TAP in the whole TAPE and in the water phase, was isocratic for the first 55 min and then changed
respectively. Vtotal and Vwater were the volumes of the gradually to solvent A (100%) over 5 min and this was
whole (100 ml) emulsion and the water phase (80 ml), maintained for 35 min, and then the initial mobile phase
respectively. composition was restored over 5 min and maintained
for an additional 10 min [22]. The air carrier flow rate
2.4.3. Long-term stability of the emulsions was 1.6 ml/min, the gain was 2, the temperature of the
drift tube was 80 ± 0.5˚C and the injection volume was
Chemical and physical stability considerations are 50 μl.
particularly important for emulsion systems, since
they are generally stored as liquids, and flocculation 3. Results and discussion
or coalescence can occur during storage, resulting in
a cream layer on the emulsion and the appearance of 3.1. Formulation investigations of the TAPE
large oil droplets or a layer of free oil [21]. In this study,
the long-term stability of the TAPE was monitored 3.1.1. Oil phase ratio and composition of the TAPE
at 25 ± 1˚C (room temperature) and 4 ± 1˚C in the
emulsion final packaging. Samples were withdrawn at The oil phase ratio and composition in emulsions
0, 1, 2, 3, 6, 9 and 12 months and assessed for physical plays an important role in the formulation. The oil
appearance, pH and droplet size distribution (intens- phase composition influences the physicochemical
wt Gaussian distribution), drug content, ζ-potential and properties and the stability of parenteral lipid emulsions
EE. [18]. In this study, a 20% (w/v) oil phase was employed
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to obtain a high drug loading efficiency. The solubility heavy flocculation was observed after three months at
of TAP in MCT is higher than that of LCT (19.17 mg/g room temperature. In addition, the oil phase consisting
vs. 9.55 mg/g), so a TAPE consisting entirely of MCT of only MCT may destabilize the emulsion system
could be loaded with more TAP than that of LCT or (i.e. Formulation D), because MCT can reduce the
LCT and MCT in a fixed ratio. However, it has been thermal tolerance of emulsions when exposed to steam
reported that MCT can destabilize the emulsion with sterilization for its solubility in water is 100-fold
respect to droplet coalescence, while LCT can increase greater than that of LCT [24]. Finally, a mixed oil phase
the viscosity of MCT and the particle size distribution composed of MCT and LCT in a ratio of 15:5 (% w/v)
of emulsions, which would increase the stability of was employed to load 6.19 mg TAP (0.4% w/v, based
emulsions during storage [23]. Moreover, LCT has on TAA) and to maintain the stability of the TAPE
been available as parenteral emulsions for clinical use during storage.
for more than 30 years, providing a better fatty acid
balance for total parenteral nutrition [24]. Hence, in this 3.1.2. The effects of lecithin type on the TAPE
study, LCT and MCT were selected as the mixed oil
phase. As mentioned above, different oil phase ratios In this study, different types of lecithin with different
and compositions in TAPE with MCT and LCT were contents of phosphatidylcholine (PC) were employed to
investigated. The results are shown in Table 1. investigate the effect on the TAPE characteristics, such
From Table 1 it can be seen that the increased as pH, mean particle size, ζ-potential and content of
proportion of LCT reduced the drug loading efficiency LPC, as shown in Table 2.
of TAPE, which was mainly due to the limited solubility The pH and width of the particle size distribution (Coeff.
of TAP in LCT (9.55 mg/g at 25˚C). On increasing the of Var’n) were the same, and the PL-100M lecithin
ratio of MCT, the TAPE particle size was reduced. This seems to be more susceptible to hydrolysis or oxidation
might be due to the higher polarity and low viscosity since the content of LPC in the TAPE was more
of MCT compared with that of LCT, resulting in than three times that of Lipoid E80 under the same
rapid and easy emulsification under the high-pressure conditions, while the LPC content was in all cases less
homogenization. However, these relatively small than the standard limit of the propofol lipid emulsion (1.2
droplets may impair the physical stability of the TAPE mg/ml). The absolute ζ-potentials differed markedly
system, since their Brownian motion was more intense and the higher the PC content, the smaller the absolute
than that of relatively larger particles, resulting in ζ-potential, showing that the absolute ζ-potential of
random collisions with flocculation or coalescence over the TAPE composed of Epikuron 170 was greater than
time [25]. As shown in the TAPE of Formulation C, that of E80 and PL-100 M, which were both purer egg
Table 1
The effects of the oil phase ratio and composition on the characterization of TAPE (with egg lecithin of PL-100M 3.0% (w/v), F68 0.2%
(w/v), and sodium oleate 0.05% (w/v), and the pH value and thermal sterilization methods for the TAPE formulation were the same as in
Section 2.2).
Physical appearance Physical appearance after
Each 100 ml TAPE LCT:MCT PSD (nm) Zeta potential (mV)
after sterilization three months at 25˚C
Formulation A 10:10 Drug precipitation ND 161.3 ± 64.67 -25.6
Formulation B 5:15 Good Good 153.4 ± 48.15 -25.8
Formulation C 2:18 Good Heavy flocculation 140.9 ± 44.52 -24.9
Formulation D 0:20 Oil floating ND 128.2 ± 35.89 -26.2
ND means not determined.
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lecithins and had a higher PC content than soybean described in 3.1.2. However, a single emulsifier of
lecithin. This could be understood by the theory that lecithin was not sufficient to maintain the stability of the
the absolute ζ-potential of lipid emulsions composed emulsion, and a combination of different co-emulsifiers
of phosphatidyl ethanolamine (PE) was greater than is needed to achieve thermal sterilization as well as the
that of emulsions composed of PC [26] and, as we long-term stability of drug-loading emulsions [24]. In
know, purified yolk lecithins are phosphatides mainly this paper, we investigated F68 and Tween 80 as co-
containing PC and PE. Moreover, soybean lecithins, emulsifiers and sodium oleate as a stabilizer. Tables
such as Epikuron 170, have a lager total interaction 3–4 show the typical formulations used in our study.
energy (Vtmax) value, and do not readily undergo A factor limiting the storage of lipid emulsions for
flocculation and coalescence [27]. In addition, parenteral administration is their physical stability, so,
emulsions with a greater negative ζ-potential (smaller the physical appearance after sterilization was chosen
than –20 mV) are better able to resist flocculation as an important index to investigate the physical
and coalescence because of the large electrostatic stability of the TAPE. Moreover, the safety of emulsion
repulsion between the emulsion droplets, which might application is to a high degree dependent on the particle
help to maintain the stability of the emulsion [28, size distribution [30, 31], especially for the produced
29]. Accordingly, soybean lecithin Epikuron 170 was fraction of larger particles (reflected by the Diameter
selected to prepare the TAPE in this study. 99%), and the ζ-potential also plays an important
role in stabilizing drug-containing emulsions through
3.1.3. Lecithin content and co-emulsifier composition of electrostatic repulsion [27]. Hence, the characterization
the TAPE of the TAPE mainly involved its physical appearance,
PSD, and ζ-potential.
Emulsifiers are another essential component of As shown in Table 3, the emulsion breaking of
lipid emulsions, and both the type and concentration Formulation I took place because 1.20% (w/v) lecithin
significantly influence the stability of these emulsions [24]. was not enough to allow good emulsification of the
A small amount of anionic lecithin can greatly reduce 20% oil phase in the TAPE, and 2.40% (w/v) lecithin
the surface tension between the oil and water phase, and produced a relatively stable emulsion but with oil
can also enhance the electrostatic repulsion between droplets floating on the emulsion, indicating that the
emulsion droplets leading to an increased absolute emulsification was still not sufficient and there was no
value of the ζ-potential of the emulsion surface [27]. In uniform dispersion in the emulsifier interfacial film.
this study, soybean lecithin (Epikuron 170) was selected The TAPE exhibited a good physical appearance after
as the main emulsifier for the TAPE formulations as thermal sterilization when the amount of lecithin was
Table 2
The effects of lecithin type on the characteristics of the TAPE (with 20% oil phase composed of MCT and LCT in a ratio of 15:5, lecithin
3.0% (w/v), F68 0.4% (w/v), and sodium oleate 0.05% (w/v); the pH value and thermal sterilization methods for the TAPE formulation
were the same as in Section 2.2) .
Parameters Lipoid E80 Epikuron 170 PL-100 M
Content of PC Not less than 80% 72% 78%
PH 7.5 7.42 7.38
PSD (nm) 185.4 ± 58.2 177.5 ± 56.4 193.2 ± 67.6
Coeff. of Var’n 0.314 0.318 0.35
ζ-potential (mV) -15.53 -28.1 -23.3
Content of LPC (mg/ml) 0.126 0.383 0.415
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as much as 2.8% (w/v) and 3.0% (w/v), but the former be reinforced with the increased lecithin, showing that
amount was still not sufficient to ensure the long-term the ζ-potential of the TAPE with 1.8% (w/v) and 3.0% (w/v)
stability of the TAPE since heavy flocculation was lecithin was increased from –16.51 mV to –28.10 mV,
observed in the Formulation IV after three months at respectively. This may be because the negative electric
room temperature, and this was a significant indicator charge of the interfacial film was increased by the
of coalescence of the TAPE. Moreover, this Table increased lecithin containing quaternary charged chlorine
also suggests that, with the increase in lecithin, the atoms [25]. Finally, 3.0% (w/v) was chosen as the
mean particle size became smaller while the standard optimum amount of lecithin in Formulation V.
deviation was not markedly changed, suggesting that Table 4 shows that F68 as a co-emulsifier plays a
the interfacial film in the TAPE was stabilized by a key role in the TAPE while Tween 80 seems to reduce
substantial amount of surfactant without causing excess the physical thermal sterilization stability since there
lecithin to be left to form micelles or liposomes. This were some visible oil droplets in the TAPE with Tween
may be because the TAP molecules, except those 80 after autoclaving at 121˚C for 10 min. It has been
encapsulated in the oil phase core, also took part in the reported that the interfacial film of emulsions can be
formation of the emulsifier interfacial film, which may altered by the incorporation of a liposoluble drug [24]
need more emulsifier to become stabilized and uniform [30]. and here the palmitate chain of the TAP may be
In addition, the electrostatic repulsive force was seen to adsorbed by or grafted on to the long-chain hydrophilic
Table 3
Effect of different amounts of soybean lecithin in the TAPE (with 20% oil phase composed of MCT and LCT in a ratio of 15:5, F68 0.2%
(w/v), and sodium oleate 0.05% (w/v), and the pH value and thermal sterilization methods for the TAPE formulation were the same as in
Section 2.2).
The amount of soybean Physical appearance ζ-potential Physical appearance after
Each 100 ml TAPE PSD (nm)
lecithin (%, w/v) after sterilization (mv) three months at 25˚C
Formulation I 1.2 Emulsion breaking ND ND ND
Formulation II 1.8 Oil floating 154.8 ± 60 –16.51 Emulsion breaking
Formulation III 2.4 Oil floating 138.2 ± 40 –18.85 Emulsion breaking
Formulation IV 2.8 Good 136.7 ± 48 –22.41 Heavy flocculation
Formulation V 3 Good 130.0 ± 50 –28.1 Good
ND means not determined.
Table 4
Effect of different ratios of F68 and Tween 80 on the characterization of TAPE with lecithin of Epikuron 170 3.0% (w/v) and a 20% (w/v)
oil phase of LCT: MCT in a ratio of 5:15, the pH value and thermal sterilization methods of the TAPE were the same as in Section 2.2.
Physical appearance Physical appearance after
Each 100 ml TAPE Tween 80: F68 PSD (nm) ζ-potential
after sterilization three months at 25˚C
Formulation VI 0.2 : 0.4 Oil floating 128 ± 35.89 –20.64 ND
Formulation VII 0.2 : 0.3 Oil floating 135.0 ± 38.06 –19.2 Emulsion breaking
Formulation VIII 0.2 : 0.2 Oil floating 169.6 ± 54.8 –20.9 Emulsion breaking
Formulation IX 0.1: 0.3 Oil floating 133.6 ± 48.22 –18.85 Oil floating
Formulation X 0: 0.4 Good 152.9 ± 50.61 –20.3 Good
Formulation XI 0: 0.5 Good 143.9 ± 46.61 –19.2 Heavy flocculation
ND means not determined.
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macro-molecules of Tween 80, producing a steric force causes the repulsion of adjacent droplets and results in
to maintain the stability of the TAPE [26, 31]. However, the formation of stable emulsions [27, 29, 32]. Floccula-
this steric force may be weakened by the heat exposure tion could be observed in the TAPE with 0.05% (w/v)
after steam sterilization because of the reduced aqueous and 0.08% (w/v) sodium oleate and their ζ-potential
solubility of Tween 80, leading to the destruction of was –20.30 mV and –25.47 mV, respectively, which
the interfacial film and oil droplets floating on the suggested that there was a lower electrostatic repulsion
surface [24, 31]. It is necessary to emphasize that the on the droplet surface compared with that of the TAPE
hypothesis that Tween 80 impairs the steam sterilization with 1.0% sodium oleate. Accordingly, 0.10% sodium
stability of emulsions applies only to the TAPE system oleate was used as a stabilizer to prepare the TAPE and
for the emulsifying interfacial film may have been to obtain a high electrostatic repulsion for long-term
altered by incorporation of the liposoluble drug of stability.
TAP, since there have been no other reports about this
phenomenon, despite similar results being obtained in 3.2. Technology Investigation of the TAPE
duplicate experiments. Moreover, by comparing these
formulations with different ratios of co-emulsifier, it 3.2.1. Effect of homogenization pressure on the PSD
is further confirmed that both the amount and type of
emulsifier affect the stability of emulsions and, if the The particle size distribution and the number of large
amount of co-emulsifier is low or even zero, phase particles are usually used to assess the physical stability
separation and final emulsion breaking can easily of emulsions and the homogenization efficiency. With
occur; also, if the amount of co-emulsifier is greater, the regard to possible toxic effects, particular attention is
excess surfactant will not only disturb the uniformity of given to the larger particles. It has been reported that
the emulsifier interfacial film, but it will also become a the mean diameter of submicron emulsions for intra-
potential safety hazard. Hence, finally, F68 was selected venous injection always lies in the 200 to 400 nm region [31]
as an important co-emulsifier for the TAPE and its and 99% of the particles (Diameter 99%) were below
optimum concentration was 0.4% (w/v) for physical 450 nm, which is safe for parental injection (seen as
thermal sterilization stability and long-term stability. the quality criterion of propofol lipid emulsion). The
Oleic acid or sodium oleate is commonly used coefficient of variation (Coeff. of Var’n) of the particles
in emulsions as a stabilizing agent capable of being represented the width of the particle size distribution
localized in the interfacial film since it can enhance in the system, and this is also a significant index of
molecular interactions and increase the electrostatic the stability and uniformity of an emulsion, i.e. 0.25 is
surface charge of droplets. In this study, different preferable for most lipid emulsions. In this experiment,
amounts of sodium oleate, from 0.05% (w/v) to 1.0% (w/v), the TAPE was homogenized using a variety of homo-
were used to investigate its effect on the characteristics genization pressures from 400 to 1200 bar, and samples
of TAPE, and the results demonstrated that there were were withdrawn after 9 cycles and mainly analyzed by
no significant differences in physical appearance after the diameter (D99%) and mean diameter.
thermal sterilization and the width of the particle size The effects of homogenization pressure on the D99%
distribution up to three months; there were also different and mean diameter of the TAPE are shown in Fig. 2.
degrees of flocculation or conglomeration except for the Applying a low homogenization pressure of 400 bar with
TAPE with 1.0% (w/v) sodium oleate, which possessed 9 cycles led to a decrease in the D99% to 493.3 nm
a relatively high ζ-potential of –32.32 mV. This increased with a mean diameter of 226.6 nm (the diameter of the
charge on the emulsion particles will ensure the long- pre-emulsion was greater than 1 μm). As the pressure
term stability of emulsions, because it has been reported increased from 800 bar to 1200 bar with the same number
that a relatively high zeta potential (>–30 mV) is needed of cycles, the mean diameter became much smaller and
in most emulsions to ensure a high-energy barrier, which more uniform. Moreover, experiments with a pressure
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1Triamcinolone acetonide submicron emulsion/Asian Journal of Pharmaceutical Sciences 2010, 5 (2): 61-73
increased up to 800 bar produced the smallest D99% perspective, this phenomenon could be also interpreted
while 900 bar failed to provide a further reduction in from the zones of droplet density distribution in the
D99%. This could be explained by assuming that the piston-gap of the homogenizer [34], and in the zone of
TAPE was forced, under high pressure, through a two- low power density, and some large particles could ‘survive’
tandem annular space and withstood two rapid disper- the homogenization cycles. On increasing the cycles, the
sion processes, wherein a hole force, shear force and surviving large particles re-dispersed with the small
collision force were created and interacted and, with homogenized particles, while the differences between
increased homogenization pressure, these forces between the two levels of particles may reduce the homogeni-
particles were enhanced, resulting in too much energy zation efficiency and uniformity of the emulsion system.
in the system and producing some very limited coale- In this experiment, there were no obvious differences
scence [33], which often resulted in instability during between 8 and 9 cycles, including the mean diameter
storage. Moreover, under the pressure of 800 bar, the (169.0 ± 44.3 nm and 167.0 ± 42.2 nm), D99% (293.2 nm
TAPE exhibited the narrowest width of size distribution and 300.3 nm, respectively) and particle size distribu-
with a relatively small coefficient of variation of 0.308 tion (coeff. of Var’n were 0.262 and 0.258) and, so,
compared with that of 600 bar (0.426) and 900 bar (0.383). an 8 cycle homogenization was finally chosen as the
This shows that only under a pressure of 800 bar could optimum number of homogenization cycles based on
the TAPE achieve a narrow distribution (145.3 ± 26.6 nm) industrial cost-effective considerations.
and a small D99% (334.7 nm). Hence, 800 bar was
chosen as the homogenization pressure for preparing 3.2.3. Sterilization method investigation of the TAPE
the TAPE.
Four different sterile methods consisting of sterilizing
3.2.2. Effect of cycle numbers on emulsion droplet size in a 100˚C rotating water bath for 45 min (I), a 117˚C
rotating autoclave water bath for 30 min (II), a 121˚C
The TAPE was homogenized using 12 homogeni- rotating autoclave water bath for 10 min (III) and for
zation cycles under a pressure of 800 bar, and samples 20 min (IV), were employed in this study to optimize
were withdrawn after 2, 4, 6, 8, 9, 10 and 12 homogeni- the thermal sterilization methods and the results are
zation cycles and analyzed by means of the particle size shown in Table 5.
diameter and the coefficient of variation and the results
600 D99%
are shown in Fig. 3.
The mean particle size decreased gradually with the 500 Mean diameter
increased number of homogenization cycles, but there
400
Particle size (nm)
was only a slight shift between the homogenized emul-
sions after 6 and 9 cycles, and then a continued decrease 300
after 9 cycles, because the increased extrinsic mechanical
200
force made the particle size smaller. However, too much
energy may also destroy the TAPE system uniformity, 100
for a pronounced enhancement occurred in the width
0
of the particle size distribution, and the coefficient of
400 600 800 900 1000 1200
variation was increased from 0.262 (167.0 ± 42.2 nm) to
Homogenization pressure (bar)
0.378 (149.0 ± 26.6 nm) with an enhanced D99% from
Fig. 2. The influence of homogenization pressure on the reduction
300.2 nm to 335.4 nm between 9 and 10 cycles. This in the TAPE. Diameter 99% and mean diameter as a function
could also be explained by the assumption that too much of the pressure using a high-pressure homogenizer (n = 3). The
energy made some homogenized droplets coalescence as components of TAPE, pH value and thermal sterilization method
of the formulation were the same as in Section 2.2.
shown in Section 3.2.1. In addition, from a technology
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1Triamcinolone acetonide submicron emulsion/Asian Journal of Pharmaceutical Sciences 2010, 5 (2): 61-73
It appears from Table 5 that the characteristics of before parenteral administration into the blood stream.
the TAPE were completely changed after sterilization, However, the TAPE could not withstand the thermal
and the mean particle sizes were greater than that of pressure for a long period, since a wider particle size
unsterilized TAPE and varied with the sterilization distribution (Coeff. of Var’n was 0.47 and 0.434) and
conditions, indicating that the emulsion particles may increased larger diameter (D99% was 457.9 nm and
be re-dispersed without destroying the interfacial film 418.1 nm) were clearly observed in TAPE sterilized by
following exposure in the thermal environment [35]. the method of II and IV, and this may be caused by the
Moreover, all these sterilization methods provided a good increased solubility of the emulsifier and co-emulsifier
physical appearance without oil droplets or phase separa- in the aqueous phase during exposure to the thermal
tion as well as good chemical stability with a high TAP steam for a long period [24]. In addition, the increase
content and encapsulation efficiency, demonstrating in LPC content of method IV was also the reason for
that the TAPE underwent thermal steam autoclaving, oxidation of the lecithin under thermal sterilization
which was a necessary terminal sterilization step for a long period compared with that of method III at
250 Mean diameter 0.45
Coeff. of Var'n 0.40
200 0.35
0.30
Particle size (nm)
150
Coeff. of Var's
0.25
0.20
100
0.15
50 0.10
0.05
0 0.00
2 4 6 8 9 10 12
Cycles numbers
Fig. 3. The influence of the number of cycles at 800 bar on the reduction of the TAPE using a high-pressure homogenizer. The mean
particle size and coefficient of variation as a function of the cycles using a high-pressure homogenizer (n = 3). The components of TAPE,
pH value and thermal sterilization method of formulation were the same as in Section 2.2.
Table 5
The effect of different thermal sterilization procedures on the characteristics of the TAPE. (with a 20% oil phase composed of MCT and
LCT in a ratio of 15:5, lecithin (Epikuron 170) 3.0% (w/v), F68 0.4% (w/v), and sodium oleate 0.05% (w/v), and the pH value for the
TAPE formulation was the same as in Section 2.2, n = 3).
Sterilization D 99% Content of LPC Drug content Drug EE
PSD (nm) Coeff. of Var’n
conditions (nm) (mg/ml) (%) (%)
Before sterilization 165.1 ± 68.2 0.413 426.3 ± 30 0.315 ± 0.073 99.4 ± 0.6 96.5 ± 0.8
Method I 181.7 ± 53.8 0.296 346 ± 23 0.415 ± 0.083 97.7 ± 2.2 96.7 ± 1.2
Method II 171.5 ± 80.6 0.47 457.9 ± 43 0.507 ± 0.104 98.5 ± 1.7 96.2 ± 1.0
Method III 184.5 ± 61.3 0.331 377 ± 46 0.383 ± 0.086 100.1 ± 0.8 96.2 ± 1.3
Method IV 167.5 ± 72.7 0.434 418.1 ± 29 0.532 ± 0.124 99.8 ± 1.3 96.9 ± 0.9
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1Triamcinolone acetonide submicron emulsion/Asian Journal of Pharmaceutical Sciences 2010, 5 (2): 61-73
the same temperature (121˚C) but for a different time No obvious effects of temperature and time on the
(20 min and 10 min, respectively). Hence, following TAPE chemical stability were observed except for
various investigations of sterile conditions, sterilizing the content of LPC, which was unavoidably produced
in a 121˚C rotating water autoclave for 10 min was by hydrolysis of the ester groups in the presence of
selected as the thermal sterilization method for the water. However, it is this evolution in the chemical
TAPE. composition that mainly leads to the destabilization of
the TAPE system which is reflected in the decreased
3.3. Physicochemical long-term stability of the pH value and absolute ζ-potential, which were caused
emulsions by the free fatty acids generated with the LPC as
well as by oil phase hydrolysis [21]. The particle size
As emulsion structures are thermodynamically distribution became wider and unstable during storage,
unstable, the shelf-life stability is a key factor in the and the coefficient of variation was increased from 0.230
product and process development and it can be predicted to 0.357 with larger particles (D99%) being increased
by the Arrhenius equation at different temperatures [36]. from 255.8 nm to 360.0 nm at 25˚C for 6 months. This
While emulsions subjected to temperature variations is because the micronized droplets undergo intense
undergo dramatic alterations and the monitored changes Brownian motion and random collisions with time at
can be erratic, a realistic stability program is needed a relatively high temperature compared with 4˚C [25],
to assess the normal shelf-life of an emulsion and leading to flocculation or some limited coalescence
this should be made on the basis of predictions of and formation of large particles. However, this of no
normal conditions undergone by the specific emulsion relevance to the product quality because the emulsions
formulation [24]. Therefore, in this study, the long-term are still extremely finely dispersed for parenteral
stability of the TAPE contained in the final packaging administration, especially those stored at 4˚C for 12
of 5 ml vials and sealed with a pulp and plastic screw months. From these experiments, it can be concluded
cap was carried out at 25 ± 1˚C (room temperature) and that the optimal storage condition for the TAPE was at
4 ± 1˚C in a refrigerator by monitoring the physical and 4 ± 1˚C in the refrigerator and the shelf life of the TAPE
chemical stability at intervals of 0, 1, 2, 3, 6, 9 and 12 was at least one year. A study of the long-term stability
months [35]. The results obtained are shown in Table 6. of the TAPE is in progress at the present time.
Table 6
Characteristics of the TAPE undergoing rotating autoclaving at 121˚C for 10 min and stored at 25˚C for 6 months and 4 ± 1˚C for 12
months (the TAPE was composed of lecithin (Epikuron 170) 3.0% (w/v) and 20% (w/v) oil phase of LCT: MCT in a ratio of 5:15, F68
0.4%, and the pH value and thermal sterilization method of the TAPE were the same as in Section 2.2, n = 3).
Characterization parameters 0 months 6 months (stored at 25˚C) Stored at 4˚C for 12 months
pH value 7.52 ± 0.08 7.08 ± 0.40 7.23 ± 0.54
PSD (nm) 162 ± 38.22 167 ± 59.73 166.4 ± 42.10
Coeff. of Var’s 0.23 0.357 0.253
D 99% (nm) 255.8 ± 23 360.0 ± 27 290.1 ± 23
ζ-Potential (mV) –35.56 ± 1.24 –28.58 ± 3.72 –24.77 ± 4.32
Physical appearance Good Little flocculation Good
Drug content (%) 99.6 ± 0.8 99.4 ± 0.9 98.12 ± 1.2
EE (%) 96.9 ± 0.72 96.5 ± 0.36 96.2 ± 0.54
Content of LPC (mg/ml) 0.383 ± 0.064 0.446 ± 0.068 0.415 ± 0.045
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1Triamcinolone acetonide submicron emulsion/Asian Journal of Pharmaceutical Sciences 2010, 5 (2): 61-73
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