Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 63 No. 5 pp.

461ñ467, 2007 ISSN 0001-6837

Polish Pharmaceutical Society

FORMULATION AND EX VIVO EVALUATION OF ROFECOXIB GEL

FOR TOPICAL APPLICATION
MALAY K. DAS* and ABDUL B. AHMED

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh 786004, India

Abstract: The potential gastrointestinal disorders associated with oral administration of rofecoxib can be avoid-
ed by delivering the drug to the inflammation site at a sustained, concentrated level over an extended period of
time. Hydroxypropylmethylcellulose (HPMC), sodium alginate and Carbopol 940 were used in an attempt to
develop topical gel formulations of rofecoxib. The effects of polymer composition on the rate of drug release
from the gel formulations were examined through cellulose membrane mounting on a Keshary-Chien diffusion
cell. The effects of initial drug concentration and viscosity on the permeation rate of rofecoxib from the gel for-
mulations were evaluated using rat epidermis at 37 ± 0.5OC. The anti-inflammatory activity of the rofecoxib gel
formulation was evaluated using the rat hind paw edema model. The gel formulation consisting of 4% w/w sodi-
um alginate-Carbopol 940 at 3:1 ratio was found to be suitable for topical application based on in vitro evalua-
tion and ex vivo permeation studies. The drug permeation rate increased with an increase of the initial drug con-
centration in gels up to 25 % w/w. An inverse relationship was observed between the in vitro drug release rate/ex
vivo permeation rate and viscosity of the gel formulations. The anti-inflammatory activity of 4% w/w sodium
alginate-Carbopol 940 gel containing 25% w/w rofecoxib in the rat hind paw edema model reveals that the drug
was delivered to the inflammation site at a controlled level over a period of 6 h. These results suggest the fea-
sibility of the topical gel formulation of rofecoxib.

Keywords: rofecoxib, sodium alginate, Carbopol 940, topical gel, anti-inflammatory activity, rat hind paw
edema model

The use of non-steroidal anti-inflammatory molecular weight of 314.36 Da and melting point in
drugs is well recognized for regional inflammatory the range of 204 ñ 208∞C can be considered ideal for
disorders such as muscle pain, osteoarthritis and transport through the skin (4). However, rofecoxib
rheumatoid arthritis. Rofecoxib, a specific COX2 has not been investigated for potential administra-
inhibitor, is one of the most potent non-steroidal tion via transdermal route except one research paper
anti-inflammatory agents. The drug was approved that reported the enhancement of skin permeation of
by FDA in the year 1999 (1, 2) for the treatment of rofecoxib using topical micro emulsion (5).
pain and inflammation associated with muscu- In the present study, transdermal gel formula-
loskeletal disorders, primary dysmenorrhea, tions for rofecoxib were prepared using HPMC,
rheumatoid arthritis and osteoarthritis. The oral sodium alginate and Carbopol 940 alone and/or in
bioavailability of rofecoxib is about 93 % and the combination, as gel forming polymers. After in vitro
steady state plasma concentration is reached at 3-4 evaluation of the gel formulations, ex vivo perme-
days with multiple dose oral administration. ation of rofecoxib from it was evaluated across rat
However, its use has been associated with a number epidermis. Further, the in vitro anti-inflammatory
of gastrointestinal disorders (3). These potential side activity of the gel formulation was studied using the
effects may be overcome by the topical administra- rat model.
tion of the drug.
The present research has been undertaken with MATERIALS AND METHODS
the aim to develop a transdermal gel formulation of
rofecoxib, which would attenuate the gastrointesti- Materials
nal related toxicities associated with oral adminis- Rofecoxib was a gift sample from Alembic
tration. The log (P) value of rofecoxib is about 1.705 Pharmaceutical Ltd. (Vadodara, India). Sodium
indicating its lipophilicity for the development of alginate, Carbopol 940 (Loba Chemie Pvt. Ltd.,
transdermal formulation. Also, rofecoxib having Mumbai, India), glycerol (Qualigens Fine

* Corresponding autor: phone: +91-373-2310052 (R), +91-373-2370254 (O), Fax: +91-373-2370323 (O); e-mail: [email protected]

461
462 MALAY K. DAS and ABDUL B. AHMED

Chemicals, Mumbai, India), methyl paraben method was validated for accuracy and precision.
(Himedia Laboratories Pvt. Ltd., Mumbai, India), When a standard drug solution was assayed repeat-
triethanolamine (Central Drug House Ltd., Mumbai, edly (n = 6), mean standard error (accuracy) and
India), Polyethylene glycol 400 (Merck Ltd., RSD (precision) were found to be 0.5% and 0.7%,
Mumbai, India), cellulose membrane (Sigma respectively.
Chemical Company, USA), sodium bromide (Loba
Chemie Pvt. Ltd., Mumbai, India) and chloroform Preparation of rofecoxib gel
(Thomas Baker Chemicals Pvt. Ltd., Mumbai, Gels were prepared by dispersing the polymers
India) were procured and used in this investigation. in a mixture of water and glycerol with methyl-
paraben as preservative and varying amount of rofe-
Animals coxib, being kept under magnetic stirring until
All experiments with animals were approved homogeneous dispersion was formed. The disper-
by the Institutional Animal Ethics Committee of sion was then neutralized and made viscous by the
Dibrugarh University, India. The Wistar male albino addition of triethanolamine. The compositions of
rats were obtained from M/S Ghosh Enterprises, different gel formations are listed in Table 1.
Kolkata, India and maintained under controlled con-
ditions of temperature as well as humidity and the In vitro evaluation
rats had free excess to water and food. The physical appearance and homogeneity of
the prepared gels were tested by visual observations.
Analytical method The spreadability of the gel formulations was deter-
The estimation of drug in the samples collect- mined at 24 h after permeation, by measuring the
ed from in vitro release and ex vivo permeation stud- spreading diameter of 1 g of gel between two hori-
ies was performed by a UV spectrophotometric zontal plates (20 cm × 20 cm) after one min. The
method. In order to generate calibration curve, an standardized weight tied on the upper plate was 125
accurately weighed amount of rofecoxib was solubi- g (6). The Voveran Emulgel (Novartis Pharma) was
lized in the solution of 40% v/v PEG 400 in water to considered as reference standard.
obtain a primary standards in the concentration The pH of the gel formulations was determined
range of 10 ñ 80 µg/mL and the calibration curve by using a pH meter (TOSHNIWAL, Model CL 54).
was obtained by measuring their absorbance at pre- The measurement was performed at 1, 30 and 60
determined λmax of 257 nm with a Hitachi U-2001 days after preparation to detect any pH fluctuation
UV-VIS spectrophotometer. The concentration of with time.
rofecoxib in test samples was calculated using the For assay of the drug in gels, rofecoxib was
linear regression equation of the calibration curve extracted from 1 g of each gel formulations with 20
(Absorbance = 0.0252 + 0.0117 × Concentration, r2 mL of methanol for 30 min. The resultant mixture
= 0.9973). The high value of correlation coefficient was filtered through membrane filter (pore size 0.45
(r2) indicates the linearity of the calibration curve mm). The absorbance of the sample was determined
and the curve did not deviate significantly from the spectrophotometrically at 257 nm (Hitachi U-2001
origin as indicated by its low value of intercept. The UV-VIS spectrophotometer) after appropriate dilu-

Table 1. Compositions of gels.

Sodium Carbopol HPMC Rofecoxib Glycerol Methyl

F.N.Code alginate (%w/w) (%w/w) (mg) (mL) paraben
(%w/w) (mg)
F1 - - 12 50 0.5 10
F2 4 - - 50 0.5 10
F3 - 2 - 50 0.5 10
F4 3 1 - 50 0.5 10
F5 3.5 0.5 - 50 0.5 10
F6 2.5 1.5 - 50 0.5 10
F7 3 1 - 10 0.5 10
F8 3 1 - 20 0.5 10
F9 3 1 - 75 0.5 10
 Rofecoxib gel for topical application 463

tion with aqueous solution of PEG 400 (40% v/v). the Keshary-Chien diffusion cell with the stratum
The concentration of rofecoxib was estimated from corneum (SC) facing the donor compartment. The
the regression equation of the calibration curve. receptor compartment was filled with aqueous solu-
The viscosity of the gel formulations was deter- tion of PEG 400 (40% v/v) and receptor phase was
mined using Brookfield viscometer with spindle no. maintained at 37 ± 0.5OC. 1 g of the gel was placed
6 at 10 rpm at the temperature of 31OC. on the SC side in the donor compartment and cov-
The in vitro drug release from gel formulations ered with aluminium foil to prevent drying out. The
was studied across cellulose membranes using amount of drug permeated was determined spec-
Keshary-Chien diffusion cell (7) with effective dif- trophotometrically at 257 nm by removing 1 mL
fusional surface area of 1.54 cm2 and a receptor cell aliquot through a hypodermic syringe fitted with a
volume of 19.5 mL. The receptor compartment was 0.22 mm membrane filter, at designated time inter-
filled with the solution of PEG 400 (40% v/v) in vals for 8 h. The volume was replenished with the
water and maintained at 37 ± 0.5OC with constant same volume of prewarmed receiver solution to
magnetic stirring. 1 g of gel was placed on the donor maintain sink conditions. Blanks are run for each set
compartment and covered with a piece of aluminium as described above with placebo gel and calculated
foil to prevent drying out. The samples (1 mL) were accordingly.
collected from the receptor compartment at prede-
termined time interval for 6 h period and replaced by In vitro anti-inflammatory activity
equal volume of fresh prewarmed receptor solution The in vitro anti-inflammatory activity of the
to maintain constant volume allowing sink condition gel formulation was performed using carrageenan
throughout the experiment. The amounts of rofecox- induced rat hind paw edema model (9). The Wistar
ib in the samples were assayed spectrophotometri- albino male rats weighing 150 ñ 210 g were fasted
cally at 257 nm against appropriate blank. overnight, but water was allowed ad libitum. The
animals were divided into three groups of four ani-
Ex vivo evaluation mals each. Group 1 (control) received placebo gel,
The abdominal hair of Wistar male albino rats, group 2 received 1.2 mg/mL of rofecoxib suspen-
weighing 150 ñ 200 g, was shaved using an electric sion in water and the group 3 received 1.2 mg/kg
razor after sacrificing with excess chloroform equivalent to rofecoxib in gel formulation.
inhalation. The abdominal skin was surgically Immediately after drug administration 0.05 mL of
removed and adhering subcutaneous fat was careful- 1% w/w solution of carrageenan was injected into
ly cleaned. The epidermis was then separated from the planter surface of the hind paw. The hind paw
dermis by soaking the full thickness skin in 2 M volume was measured at different time intervals for
sodium bromide solution in water for 6 ñ 8 h (8). 6 h after carrageenan treatment using a plethysmo-
The epidermis was thoroughly washed with water, graph. The percent inhibition in hind paw edema
dried at 25% RH, wrapped in aluminium foil and volume was calculated using the following formula
stored in freeze until further use. and compared with those recorded for control group.
For ex vivo permeation studies, skins were Anti-inflammatory activity (%) = (1 ñ D/C) × 100
allowed to hydrate for 1 h before being mounted on where D is the change in paw volume in the treated

Table 2. Physicochemical evaluation of gel formulations.

Spreading % Rofecoxib Physical pH

F.N.Code diameter after content appearance Homogeneity (mean ± SD)
1 min (mm) (mean ± SD)
F1 72 86.76 ± 5.23 White Homogeneous 6.70 ± 0.03
F2 64 87.28 ± 4.25 Opaque Homogeneous 7.09 ± 0.04
F3 48 90.16 ± 6.52 White Homogeneous 6.56 ± 0.03
F4 54 88.03 ± 5.01 Opaque Homogeneous 6.35 ± 0.04
F5 52 87.03 ± 4.04 Opaque Homogeneous 6.39 ± 0.01
F6 60 86.87 ± 3.58 Opaque Homogeneous 6.54 ± 0.02
Voveran
Emulgel 53 ñ Opaque Homogeneous 6.90 ± 0.05
(Novartis
Pharma)
464 MALAY K. DAS and ABDUL B. AHMED

Table 3. Release parameters of rofecoxib from F1 to F6 gel formulations through cellulose membrane.

Amount release Release rate Best fit regression

F.N.Code at 6 h (µg/cm2) (µg/cm2/h) equation for r2
(mean ± SD) (mean ± SD) release plot
F1 1857.10 ± 80.25 491.73 ± 24.19 Q = 491.73 t +141.11 0.9216
F2 2912.96 ± 101.52 579.08 ± 23.24 Q = 579.08 t + 132.02 0.9870
F3 2149.01 ± 88.26 510.21 ± 22.99 Q = 510.21 t + 118.64 0.9257
F4 3016.44 ± 104.23 632.54 ± 25.83 Q = 632.54 t + 181.57 0.9776
F5 1750.72 ± 78.55 512.50 ± 24.33 Q = 512.50 t + 88.79 0.8825
F6 1635.01 ± 85.66 487.97 ± 15.69 Q = 487.97 t + 110.52 0.8588

Table 4. Permeation parameters of rofecoxib from various gel formulations across rat epidermis.

Amount permeated Best fit regression

J (µg/cm2/h) TL (h) KP (cm2/S × 10-6)
F.N.Code at 8 h (µg/cm2) equation for r2
(mean ± SD) (mean ± SD) (mean ± SD)
(mean ± SD) permeation plot
F2 1748.96 ± 78.35 229.28 ± 10.12 0.41 ± 0.12 6.36 ± 0.21 Q = 229.28 t ñ 80.19 0.9932
F3 1397.11 ± 84.55 179.14 ± 8.52 0.37 ± 0.03 4.97 ± 0.15 Q = 179.14 t ñ 55.44 0.9933
F4 1948.32 ± 91.45 254.63 ± 10.98 0.40 ± 0.01 7.07 ± 0.42 Q = 254.63 t ñ 107.16 0.9934
F7 453.66 ± 40.12 88.12 ± 5.09 0.31 ± 0.02 2.45 ± 0.12 Q = 88.12 t ñ 43.39 0.9636
F8 1005.80 ± 70.15 141.31 ± 5.63 0.34 ± 0.03 3.92 ± 0.08 Q = 141.31 t ñ 11.32 0.9795
F9 1732.36 ± 83.25 249.10 ± 10.57 0.43± 0.08 7.21 ± 0.17 Q = 249.10 t ñ 120.18 0.9848

Table 5: Percent inhibitions of hind paw edema.

Formulations Percent inhibition

1h 2h 3h 4h 5h 6h
Rofecoxib oral 16.66 45.00 52.21 65.51 63.13 62.89
Rofecoxib gel 9.44 35.10 46.34 55.45 56.22 58.93

group and C is the change in paw volume in the con- showed good homogeneity and spreadability. The
trol group. physical appearance of the gel formulations was
white or opaque in nature. The drug content of the
Data and statistical analysis gel formulations was in the range of 86.76 ± 5.23 to
The steady state flux (J, µg/cm2/h) was calcu- 90.16 ± 6.52%, showing content uniformity. The pH
lated from the slope of the linear plot of the cumula- of the gel formulations was in the range of 6.35 ±
tive amount permeated per unit area (µg/cm2) as a 0.04 to 7.09 ± 0.04, which lies in the normal pH
function of time (t, h). The lag time (tL, h) was deter- range of the skin and would not produce any skin
mined from the x-intercept of the slope at the steady irritation. There was no significant change in pH
state. The permeability coefficient (KP, cm2/S) was values (varied from 0.01 to 0.16) as a function of
calculated from the flux and donor drug concentra- time for all formulations. The physicochemical
tion. properties of the prepared gel formulations were in
Data are represented as mean ± SD (n = 3). good agreement with those of a marketed product
Statistical comparisons were made using Studentís namely Voveran Emulgel from Novartis Pharma.
t-test at a significance level of p < 0.05 using MS- The viscosity of the gel formulations generally
Excel software. reflects its consistency. The consistency of F2 (4%
w/w sodium alginate), F3 (2% Carbopol 940) and F4
RESULTS AND DISCUSSION (4% sodium alginate-Carbopol 940 at 3:1 ratio) gel
formulations can be ranked according to their viscos-
In vitro evaluation ity values as follows: F3 (viscosity 89000 cps) > F2
The physicochemical properties of the gel for- (viscosity 28000 cps) > F4 (viscosity 21000 cps).
mulations are shown in Table 2. From the results it The experiments for in vitro release of rofecox-
is clearly evident that all the gel formulations ib from gel formulations through cellulose mem-
 Rofecoxib gel for topical application 465

brane were carried out to select appropriate polymer relationship [r2 > 0.9 (0.94 ñ 0.97)] was obtained
composition for gel formulation having suitable between the amount released and the square root of
consistency for topical application. Figure 1 depicts time as proposed by the Higuchiís theory (10), indi-
the release profile of rofecoxib from various gel for- cating the diffusion controlled mechanism of drug
mulations across the cellulose membrane. The release. The results reveal that the total amount of
release parameters are tabulated in Table 3. A linear drug release at 6 h for the formulations F1 to F6 was

F1 (12% w/w HPMC gel), F2 (4% w/w sodium alginate gel), F3 (2% w/w Carbopol 940 gel), F4 (4% w/w sodium alginate-Carbopol 940
at 3:1 ratio gel), F5 (4% w/w sodium alginate-Carbopol 940 at 7:1 ratio gel), F6 (4% w/w sodium alginate-Carbopol 940 at 5:3 ratio gel)

Figure 1. Release profiles of rofecoxib from various gel formulations across cellulose membrane. Bar represents mean ± S.D (n = 3).

F2 (4% w/w sodium alginate gel), F3 (2% w/w Carbopol 940 gel), F4 (4% w/w sodium alginate-Carbopol 940 at 3:1 ratio gel)

Figure 2. Permeation profiles of rofecoxib from various gel formulations across rat epidermis. Bar represents mean ± S.D (n = 3).
466 MALAY K. DAS and ABDUL B. AHMED

Figure 4. Change in edema volume with rofecoxib oral, placebo

gel and rofecoxib gel after carragenan treatment. Bar represents
Figure 3. Effect of initial drug concentration on permeation of mean ± S.D (n = 4).
rofecoxib from sodium alginate-Carbopol 940 (3:1 ratio) gel
across rat epidermis. Bar represents mean ± S. D (n = 3)

dependent on the nature of the polymer used and

viscosity, as well as the consistency of the formula-
tions. The drug release was inversely proportional to
the viscosity of the gel formulations. The gel formu-
lations consisting of the mixture of sodium alginate
and Carbopol showed good physicochemical char-
acteristics for topical application. The drug release
increased significantly (p < 0.05) when the amount
of Carbopol in the gel formulation was increased
from 0.5% (formulation F5) to 1% w/w (formulation
F4), beyond which a significant decrease (p < 0.05)
in drug release was observed (formulation F6). The
maximum drug release was observed with the for-
mulation F4 (4% w/w sodium alginate-Carbopol Figure 5. Percent inhibition of hind paw edema after oral adminis-
940 at 3:1 ratio) followed by the formulation F2 (4% tration of rofecoxib and application of rofecoxib gel.

w/w sodium alginate) and F3 (2% w/w Carbopol)

(Table 3). However, no statistically significant dif-
ference in drug release was observed between F2
and F4 (p < 0.05). It may be due to the softness and mechanism. The various permeation parameters are
less viscous nature of the formulations. The rank tabulated in Table 4. The steady state flux was
order of the various gel formulations based upon observed after a small lag time in the range of 0.31
their maximum drug release is F4 > F2 > F3 > F1 > ñ 0.43 h. An inverse relationship was observed
F5 > F6. Based on the physicochemical properties between permeation rate and the viscosity of the gel
and drug release, the formulation F4 was found to be formulation. The results are presented in Table 4.
suitable for topical application. Figure 3 depicts the effect of initial drug concentra-
tion on permeation of rofecoxib from 4% w/w sodi-
Ex vivo evaluation um alginate-Carbopol 940 gels at 3:1 ratio. The
Figure 2 and 3 depict the ex vivo skin perme- cumulative amounts permeated at 8 h were 453.66 ±
ation profile of rofecoxob from gels across rat epi- 40.12, 1005.8 ± 70.15, 1948.32 ± 91.45 and 1732.36
dermis. The skin permeation profile showed the ± 83.25 µg/cm2 from the gel formulations containing
same pattern as that of the in vitro release profile 5% (F7), 10% (F8), 25% (F4) and 37.5% (F9),
across the cellulose membrane. A linear relationship respectively (Table 4). The results reveal that there
[r2 > 0.9 (0.96 ñ 0.99)] was observed between the is a significant increase in permeation flux (p < 0.05)
cumulative amount permeated and time, indicating with increasing the drug concentration from 5% to
zero order permeation kinetics and the permeation 25% w/w, further that no significant increase in per-
of rofecoxib was based on diffusion controlled meation rate was observed (p > 0.05) (formulation
 Rofecoxib gel for topical application 467

F9 containing 37.5% w/w drug). This could be of this gel formulation in rat hind paw edema model
attributed to the increased viscosity of the gel for- reveals that rofecoxib was delivered to the inflam-
mulations at higher drug concentration. mation site at a controlled level over a period of 6 h.
These results suggest the feasibility of the topical
In vitro anti-inflammatory activity gel formulation of rofecoxib.
Figure 4 represents the change in edema vol-
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