4 Thai J. Pharm. Sci.

32 (2008) 4-9

Original article

Stability evaluation of celecoxib nanoemulsion containing Tween 80

F. Shakeel1*, S. Baboota2, A. Ahuja2, J. Ali2, M.S. Faisal2 and S. Shafiq3

1Department of Pharmaceutics, Faculty of Pharmacy, Al-Arab Medical Sciences University, Benghazi, Libya
2Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India
3New Drug Delivery System (NDDS), Zydus Cadila Healthcare Ltd., Ahemdabad, India.
*Corresponding author: Tel: +00218-924018245 E-mail address: [email protected]

Abstract:

The aim of the present study was to evaluate the stability of celecoxib (CXB) using novel
nanoemulsion formulation. Optimized nanoemulsion formulation of CXB was prepared by low energy
emulsification method. Stability studies were performed for the period of 3 months. Droplet size, viscosity
and refractive index (RI) were determined during storage. Shelf life of nanoemulsion formulation was also
determined by accelerated stability testing. It was found that droplet size viscosity and RI were slightly
increased at refrigerator and room temperature. The changes in these parameters were not significant (p ≥ 0.05).
The shelf life of nanoemulsion formulation was found to be 2.73 years at room temperature. These results
indicated that stability of CXB can be enhanced in nanoemulsion formulation using Tween 80 as surfactant.

Keywords: Celecoxib; Nanoemulsion; Stability; Tween 80

F. Shakeel et al. 5

Introduction used in the study were of analytical reagent grade.

Nanoemulsions are thermodynamically stable,
Preparation of CXB nanoemulsion
transparent (or translucent) dispersions of oil and water
stabilized by an interfacial film of surfactant and Various nanoemulsions of CXB were prepared by
cosurfactant molecules having the droplet size less low energy emulsification method (aqueous phase titration
than 100 nm [1-3]. Nanoemulsions have a higher method). Detail description of their preparation, physical
solubilization capacity than simple micellar solutions stability, characterization and optimization is given in
and their thermodynamic stability offers advantages our previously published article [1]. Optimized nanoemulsion
over unstable dispersions, such as emulsions and was prepared by dissolving 2% w/w of CXB in 10% w/w
suspensions, because they can be manufactured with mixture of Sefsol-218 and Triacetin (1:1). Then 50% w/w
little energy input (heat or mixing) and has a long shelf mixture of Tween 80 and Transcutol-P were added slowly
life [4-6]. Nanoemulsions also present advantages over in oil phase. Then slow addition of distilled water was
other nano or micro carriers, namely higher storage done to get the final preparation 100% w/w.
stability, lower preparation cost, good production feasibility,
thermodynamic stability, absence of organic solvents Characterization of nanoemulsion
and no need of intensive sonication [7-8]. Stability of a Droplet size distribution of the nanoemulsion was
dosage form refers to the chemical and physical integrity determined by photon correlation spectroscopy (PCS),
of the dosage unit and when appropriate, the ability of using a Zetasizer 1000 HS (Malvern Instruments, UK).
the dosage unit to maintain protection against Light scattering was monitored at 25 ÌC at a scattering
microbiological contamination. An ideal drug product angle of 90 Ì . The viscosity of the nanoemulsion was
must be thoroughly characterized physically, chemically determined using Brookfield DV III ultra V6.0 RV cone
and microbiologically at the start of study and throughout and plate rheometer (Brookfield Engineering Laboratories,
the intended shelf life period [9]. Stability of drug product Inc, Middleboro, MA) using spindle # CPE40 at 25±0.3 Ì
is one of the problems associated in the development C. Refractive index of nanoemulsion formulation was
of emulsions, microemulsions and nanoemulsions. determined using an Abbes type refractrometer (Precision
Nanoemulsions have been known to enhance the physical Standard Testing Equipment Corporation, India).
as well as chemical stability of drugs [1-2, 10]. Therefore,
the aim of the present study was to evaluate the stability Stability studies
parameters of celecoxib (CXB) using nanoemulsion Stability studies on optimized nanoemulsion were
formulation. Nanoemulsion formulation was prepared performed by keeping the sample at refrigerator
using nonirritant and pharmaceutically acceptable temperature (4 ÌC) and room temperature (25 ÌC). These
ingredients. studies were performed for the period of 3 months.
The droplet size, viscosity and RI were determined using
Materials and methods methods described above during storage. Accelerated
Materials stability studies were also performed on optimized CXB
CXB was a kind gift sample from Ranbaxy Research nanoemulsion. Three batches of formulations were taken
Labs (India). Propylene glycol mono caprylic ester (Sefsol in glass vials and were kept at accelerated temperature
218) was gifted from Nikko Chemicals (Japan). Diethylene of 30 ÌC, 40 ÌC, 50 ÌC and 60 ÌC at ambient humidity. The
glycol monoethyl ether (Transcutol-P) was gift sample samples were withdrawn at regular intervals of 0, 1, 2
from Gattefosse (France). Glycerol triacetate (Triacetin) and 3 months and were analyzed for drug content by
was purchased from E-Merck (India). Tween 80 was stability-indicating HPLC method at a wavelength of
purchased from Sigma Aldrich (USA). All other chemicals 250 µm [11]. Zero time samples were used as controls.
6 Thai J. Pharm. Sci. 32 (2008) 4-9

Analysis was carried out at each time interval by taking [9]. Therefore optimized nanoemulsion formulation was
50 µl of each formulation and diluting it to 5 ml with characterized for droplet size, viscosity and RI for the
methanol and injecting into the HPLC system at 250 nm. period of three months. During stability studies droplet
In addition, samples of pure oil (combination of Sefsol size, viscosity and RI were determined at temperatures
218 and Triacetin), pure surfactant and cosurfactant (Smix) of 4 ÌC and 25 ÌC. These parameters were determined at
were run separately to check there was no interference 0, 1, 2 and 3 months. It was found that droplet size
of the excipients used in the formulations. viscosity and RI were slightly increased at both
The amount of drug degraded and the amount temperatures (Table 1). These parameters were compared
remaining at each time interval was calculated. Order of for statistical significance by one-way analysis of variance
degradation was determined by the graphical method. (ANOVA) followed by Tukey-Kramer multiple comparisons
Degradation rate constant (K) was determined at each test using GraphPad Instat software (GraphPad Software
temperature. Arrhenius plot was constructed between Inc., CA, USA). The changes in these parameters
log K and 1/T to determine the shelf life of optimized were not significant (p ≥ 0.05). These results indicated
nanoemulsion formulation. The degradation rate constant that optimized formulation is stable and suitable for
at 25 ÌC (K25) was determined by extrapolating the transdermal delivery of CXB. For accelerated stability
value of 25 ÌC from Arrhenius plot. The shelf life (T0.9) studies, samples were withdrawn at regular intervals of
for each formulation was determined by using the 0, 1, 2, and 3 months. The samples were analyzed for
formula: their drug content by HPLC analysis at a wavelength
of 250 nm [11]. The amount of CXB degraded and
remaining in nanoemulsion formulation was determined
T0.9 = 0.1052
K25 at each time interval. The degradation of CXB was very
slow at each temperature that could be stability of CXB
Results and discussion in the form of nanoemulsion.
Stability of a dosage form refers to the chemical Observations are given in the Table 2. Order of
and physical integrity of the dosage unit and when degradation was determined by graphical method at
appropriate, the ability of the dosage unit to maintain each temperature. The order of degradation was found
protection against microbiological contamination. An ideal to be first order (Figure 1). The correlation coefficients
emulsion formulation must be thoroughly characterized of first order degradation were significant as compared
physically, chemically and microbiologically at the start to correlation coefficients of zero order degradation at
of study and throughout the intended shelf life period each temperature as shown in Figures 1 and 2 (p<0.05).

Table 1 Droplet size, viscosity and RI of optimized nanoemulsion formulation during storage
Time Temp Droplet size Viscosity mean RI ± SDb
(months) ( ÌC) mean ± SD (nm)a ± SD (cps)
a

0 4.0 ± 0.5 34.64 ± 3.74 19.57 ± 1.99 1.402 ± 0.006

1 4.0 ± 0.5 35.12 ± 4.04 19.62 ± 1.94 1.404 ± 0.009
2 4.0 ± 0.5 35.34 ± 4.13 19.79 ± 2.11 1.405 ± 0.008
3 4.0 ± 0.5 35.58 ± 4.34 20.18 ± 2.33 1.407 ± 0.007
0 25 ± 0.5 34.64 ± 3.74 19.57 ± 1.99 1.402 ± 0.006
1 25 ± 0.5 35.49 ± 4.38 20.06 ± 2.05 1.406 ± 0.010
2 25 ± 0.5 36.12 ± 4.51 20.38 ± 2.26 1.407 ± 0.012
3 25 ± 0.5 36.33 ± 4.84 20.63 ± 2.49 1.408 ± 0.014
a
Mean ± SD, n= 3, bMean ± SD, n = 6
F. Shakeel et al. 7

2.002
2 30oC R2 = 0.9792
1.998

Log % drug remained

1.996 R2 = 0.9692
o
1.994 40 C
1.992
1.99 50oC R2 = 0.9869
1.988
1.986
60oC R2 = 0.9779
1.984
1.982
0 0.5 1 1.5 2 2.5 3 3.5
Time (months)

Figure 1 First order degradation kinetics of CXB from nanoemulsion formulation at different temperatures

Table 2 Degradation of optimized nanoemulsion formulation

Time Temp Conc. found Conc. degraded % Log %
(months) ( ÌC) (mg) (mg) Remained remained
0 30 ± 0.5 20.00 0.00 100.00 2.0000
1 30 ± 0.5 19.95 0.05 99.75 1.9989
2 30 ± 0.5 19.93 0.07 99.65 1.9984
3 30 ± 0.5 19.89 0.11 99.45 1.9976
0 40 ± 0.5 20.00 0.00 100.00 2.0000
1 40 ± 0.5 19.91 0.09 99.55 1.9980
2 40 ± 0.5 19.85 0.15 99.25 1.9967
3 40 ± 0.5 19.81 0.19 99.05 1.9958
0 50 ± 0.5 20.00 0.00 100.00 2.0000
1 50 ± 0.5 19.84 0.16 99.20 1.9965
2 50 ± 0.5 19.73 0.27 98.65 1.9940
3 50 ± 0.5 19.52 0.48 97.60 1.9894
0 60 ± 0.5 20.00 0.00 100.00 2.0000
1 60 ± 0.5 19.75 0.25 98.75 1.9945
2 60 ± 0.5 19.44 0.56 97.20 1.9876
3 60 ± 0.5 19.31 0.69 96.55 1.9847

Table 3 Observation table for calculation of shelf life of nanoemulsion formulation

Temp. Slope K x 10-3 Log K Absolute 1/T x103

( ÌC) (month-1) Temperature
30 -0.0008 1.8424 -2.7346 303.00 3.300330
40 -0.0014 3.2042 -2.4943 313.00 3.194488
50 -0.0034 7.8302 -2.1062 323.00 3.095975
60 -0.0053 12.2059 -1.9134 333.00 3.003003
25 - 3.2033 -2.4944 298.00 3.355704
8 Thai J. Pharm. Sci. 32 (2008) 4-9

20.1
20 30oC
R2 = 0.9761
Drug remained (mg)
19.9
R2 = 0.9689
19.8
40oC
19.7
19.6 R2 = 0.9856
50oC
19.5
19.4
60oC R2 = 0.9776
19.3
19.2
0 0.5 1 1.5 2 2.5 3 3.5
Time (months)

Figure 2 Zero order degradation kinetics of CXB from nanoemulsion formulation at different temperatures

0
2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35
-0.5

-1
y = -2.8794x + 6.7535
Log K

-1.5 R2 = 0.9852
-2

-2.5

-3
1/T x 103

Figure 3 Arrhenius plot between Log K and 1/T for nanoemulsion formulation
F. Shakeel et al. 9

Therefore for first order degradation, Log % of drug References

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degradation rate constant (K) was calculated from the Design development and evaluation of novel nanoemulsions
slope of the curve at each temperature. Observations formulations for transdermal potential of celecoxib, Acta
are given in the Table 3. The degradation rate constant Pharm. 8: 316-332 (2007).
was calculated by the formula [2] S. Shafiq, F. Shakeel, S. Talegaonkar, F.J. Ahmad, R.K. Khar,
and M. Ali. Development and bioavailability assessment of
-K ramipril nanoemulsion formulation, Eur. J. Pharm. Biopharm.
Slope = 66: 227-242 (2007).
2.303
[3] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, M. Aqil, and S.
Shafiq. Nanoemulsions as vehicles for transdermal delivery
The effect of temperature on the degradation was studied of aceclofenac, AAPS Pharm. Sci. Tech. 8: E104 (2007).
by plotting log K v/s 1/T. (Figure 3). The value of K at [4] S. Baboota, A. Alazaki, K. Kohli, J. Ali, N. Dixit, and F.
25 ÌC (K25) was obtained by extrapolation of the plot Shakeel. Development and evaluation of a microemulsion
and shelf life was then calculated by substituting K25 formulation for transdermal delivery of terbenafine, PDA J.
in the following equation: Pharm. Sci. Tech. 61: 276-285 (2007).
[5] S. Shafiq, F. Shakeel, S. Talegaonkar, F.J. Ahmad, R.K.
0.1052 Khar, and M. Ali. Design and development of oral oil in
T0.9 = water ramipril nanoemulsion formulation: In vitro and in
K25
vivo assessment, J. Biomed. Nanotech. 3: 28-44 (2007).
Where T0.9 is the time required for 10 % drug degradation [6] S. Shafiq, F. Shakeel, S. Talegaonkar, J. Ali, S. Baboota, A.
Ahuja, R.K. Khar, and M. Ali. Formulation development
and is referred to as shelf life. The shelf life of optimized and optimization using nanoemulsion technique: a technical
nanoemulsion formulation was found to be 2.73 years. note, AAPS Pharm. Sci. Tech. 8: E28 (2007).
[7] D. Paolino, C.A. Ventura, S. Nistico, G. Puglisi, and M. Fresta.
Conclusion Lecithin microemulsions for the topical administration of
ketoprofen: percutaneous absorption through human skin
The droplet size, viscosity and RI of optimized and in vivo human skin tolerability, Int. J. Pharm. 244:
nanoemulsion formulation were not significantly changed 21-31 (2002).
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that prepared nanoemulsion was physically stable. Celecoxib nanoemulsion for transdermal drug delivery:
The degradation of CXB after 3 months of storage was Characterization and in vitro evaluation, J. Disp. Sci. Tech.,
In press.
very low in the nanoemulsion formulation. The degradation
[9] A.G. Floyd. Top ten considerations in the development of
of CXB was increased by increasing the temperature. parenteral emulsions, Pharm. Sci. Tech. Today 2: 134-143
The shelf life of nanoemulsion formulation was found (1999).
to be 2.73 years at room temperature. Overall these [10] F. Shakeel, S. Baboota, A. Ahuja, J. Ali, and S. Shafiq.
results indicated that stability of CXB can be enhanced Skin permeation mechanism of aceclofenac using novel
in nanoemulsion formulation using Tween 80 as surfactant. nanoemulsion formulation, Pharmazie, In press.
[11] S. Baboota, F. Shakeel, A. Ahuja, J. Ali, S. Ahmed, and S.
Shafiq. Development and validation of a stability-indicating
Acknowledgement HPLC method for analysis of celecoxib in bulk drug and
The authors are thankful to University Grant microemulsion formulation, Acta Chromat. 18: 116-129
Commission (UGC), New Delhi, India for providing (2007).
financial support to this project.