Enhanced transdermal delivery of ketoprofen from bioadhesive gels.

Abstract

The aim of this study was to evaluate and compare the in vitro and in vivo transdermal potential of bioadhesive gels of ketoprofen by using gelling polymers like sodium carboxymethylcellulose, xanthan gum, poloxamer 407 and carbopol 934P as bioadhesive polymer with and without penetration enhancer (oleic acid). The effect of oleic acid as a penetration enhancer was examined when it was added to the bioadhesive formulations. Gels were evaluated for bioadhesive force and viscosity. To study the in vitro potential of these formulations, permeation studies were performed with Franz diffusion cell using excised rat abdominal skin. Carrageenan induced rat paw edema model was used to investigate their in vivo performance. The commercial formulation of ketoprofen was used as a reference formulation. The in vitro permeation studies indicate that ketoprofen bioadhesive gel of poloxamer 407 with penetration enhancer was superior to gels of sodium carboxymethylcellulose and xanthan gum with penetration enhancer (oleic acid). The permeation rate of ketoprofen from poloxamer 407 based bioadhesive gel with 15% v/w penetration enhancer was higher (rat abdominal skin flux = 0.421 +/- 0.032 mg/cm(2)/h) than the permeation rate of sodium carboxymethylcellulose and xanthan gum based bioadhesive gel with 15% v/w penetration enhancer. In the paw edema test poloxamer 407 based bioadhesive gel with 15% v/w penetration enhancer showed the best permeation and effectiveness. The in vitro and in vivo studies showed that bioadhesive gels of ketoprofen could be used for effective therapy.

Full text

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Pak. J. Pharm. Sci., Vol.22, No.2, April 2009, pp.193-198
193
ENHANCED TRANSDERMAL DELIVERY OF KETOPROFEN
FROM BIOADHESIVE GELS

S SINGH*, B GAJRA, M RAWAT AND M S MUTHU**
Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi-221005, India
**Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University,
Varanasi-221005, India

ABSTRACT
The aim of this study was to evaluate and compare the in vitro and in vivo transdermal potential of bioadhesive
gels of ketoprofen by using gelling polymers like sodium carboxymethylcellulose, xanthan gum, poloxamer 407
and carbopol 934P as bioadhesive polymer with and without penetration enhancer (oleic acid). The effect of oleic
acid as a penetration enhancer was examined when it was added to the bioadhesive formulations. Gels were
evaluated for bioadhesive force and viscosity. To study the in vitro potential of these formulations, permeation
studies were performed with Franz diffusion cell using excised rat abdominal skin. Carrageenan induced rat paw
edema model was used to investigate their in vivo performance. The commercial formulation of ketoprofen was
used as a reference formulation. The in vitro permeation studies indicate that ketoprofen bioadhesive gel of
poloxamer 407 with penetration enhancer was superior to gels of sodium carboxymethylcellulose and xanthan
gum with penetration enhancer (oleic acid). The permeation rate of ketoprofen from poloxamer 407 based
bioadhesive gel with 15% v/w penetration enhancer was higher (rat abdominal skin flux = 0.421 ± 0.032
mg/cm2/h) than the permeation rate of sodium carboxymethylcellulose and xanthan gum based bioadhesive gel
with 15% v/w penetration enhancer. In the paw edema test poloxamer 407 based bioadhesive gel with 15% v/w
penetration enhancer showed the best permeation and effectiveness. The in vitro and in vivo studies showed that
bioadhesive gels of ketoprofen could be used for effective therapy.

Keywords: Ketoprofen; bioadhesive gel; permeation; rat skin; in vivo study.

INTRODUCTION

Ketoprofen (KP) is a potent non-steroidal anti-
inflammatory drug that inhibits prostaglandin synthetase
cyclooxygenase. KP is widely used for acute and long-
term management of rheumatoid arthritis. However, the
oral administration of this drug is usually accompanied by
severe gastric and duodenal irritation, and because of its
short half-life of 1.1-4 h, it requires frequent oral dosage
(Lanza et al., 1998; El-kattan et al., 2000).

Percutaneous absorption of KP would be a possible
alternative offering distinct advantages, such as
elimination of gastric and duodenal irritation and
delivering the drug directly to the inflamed site and
thereby producing prolonged
concentrations.

Percutaneous drug delivery has some advantages of
providing the controlled delivery of drugs. In case of their
application such as ointments, creams, it is difficult to
expect their effects, because wetting, movement and
contacting easily remove them. There is a need to develop
the new formulations that have suitable bioadhesion. The
percutaneous administration of bioadhesive gels has good
accessibility and can be applied, localized and removed
easily. Because of its excellent accessibility, self-
therapeutic local
placement of a dosage form is possible (Shin and Cho,
2006).

But skin is a natural barrier for transdermal administration
and only a few drugs can penetrate the skin easily and in
sufficient levels to be effective (Barry 2001; Moser et al.,
2001).

Therefore, in recent years, penetration enhancers such as
hydrogenated soybean phospholipids, ethanol, alcohols
with long carbon chains (C8 to C14), n-octanol and cyclic
monoterpenes, nonionic surfactants, propylene glycol and
isopropyl myristate have been used in many studies to
increase the percutaneous absorption of drugs (Nishihata
et al., 1988; Parsaee et al., 2002; Ho et al., 1994; Iwasa et
al., 1991; Santoyo et al., 1995). Membranes from rats,
mice, pigs, guinea pigs, snakes, rabbits, and humans as
well as synthetic membranes have been used for these
drug diffusion studies (Nair and Panchagnula, 2004;
Tokudome and Sugibayashi, 2003).

Recently, bioadhesive gels have been developed for
piroxicam, triamcinolone
griseofulvin and methotrexate (Santoyo et al., 1995; Shin
et al., 1999; Shin et al., 2000; Shin et al., 2003; Vlachou
et al., 1992; Lu and Jun 1998). These gels were studied
for the bioadhesive character and diffusion parameters.
acetate, benzocaine,
*Corresponding author: e-mails: drsanjaysingh@rediffmail.com; muthubits@rediffmail.com

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Enhanced transdermal delivery of ketoprofen from bioadhesive gels
Pak. J. Pharm. Sci., Vol.22, No.2, April 2009, pp.193-198
194
Oleic acid (OA) is considered to have good permeation
enhancing property by acting as a lipid disrupting agent
that increases the fluidity of stratum corneum lipid by
increasing the formation of water channels (Naik et al.,
1995; Fang et al., 2003; Francoeur et al., 1990).

Carbopols are excellent bioadhesive polymers but they
have very low pH in the range of 2.5-3.0 (1% aqueous
solution) (Ahuja et al., 1997). If used alone, they may
cause irritation following topical application due to there
low pH. Its irritant properties can be reduced by
combining it with other non-irritant bioadhesive
polymers.

Therefore, it was proposed to develop a topical
bioadhesive gel systems of KP with and without
penetration enhancer (OA). Polymers used are sodium
carboxymethylcellulose (NaCMC), xanthan gum (XG),
poloxamers 407 (PL407) and carbopol 934P (CB934P).

Bioadhesion properties, viscosity studies and in vitro
permeation of these selected gels were studied through the
excised full thickness rat abdominal skin. The prepared
gels were compared with the conventional marketed gel
formulation. Furthermore, pharmacodynamic studies of
gels were evaluated for anti-inflammatory activity on a
carrageenan-induced rat paw edema model. This study
aimed to enhance topical penetration of KP from different
bioadhesive gels and to compare with the commercial
formulation available.

EXPERIMENTAL

Materials
Ketoprofen (KP) was obtained as gift sample from
Themis Labs Pvt. Ltd., Mumbai, India. Sodium
carboxymethylcellulose (NaCMC) and oleic acid (OA)
were kindly supplied by s.d fine-chem. Ltd, (Mumbai).
Xanthan gum (XG) and carbopol 934P (CB934P) were
supplied from Loba Chemie Pvt. Ltd., (Mumbai).
Poloxamer 407 (PL407) was supplied from BASF India
Ltd., (Mumbai). All other chemicals used were of
analytical grade.

Preparation of ketoprofen gel with NaCMC and XG
The required amount of gelling polymer (NaCMC or XG)
and CB934P were weighed (table 1). Weighed polymers
were added slowly in the beaker containing distilled water
(40 ml) with continuous stirring at 400-600 rpm. The
mixture was stirred continuously for 1 h until it forms a
clear gel. Accurately weighed KP was dissolved in 30 ml
of ethanol and the ethanolic solution of drug was added
slowly with stirring (400-600 rpm) in the previously
prepared polymer gel. Penetration enhancer OA was
added with stirring. The final quantity was made up to
100 g with distilled water. The prepared gel was kept for
24 h for complete polymer desolvation.

Preparation of ketoprofen gel with PL407
PL407 possesses reverse thermal gelling property and
therefore the gel containing PL407 was prepared by cold
method (Schmolka 1972). The required amount of PL407
and CB934P were weighed (table 1). Weighed PL407 was
added slowly in the 5oC cold distilled water (20 ml) with
stirring (400-600 rpm) and CB934P was separately
dissolved in normal distilled water (5 ml) and stirred.
Then CB934P solution was added to PL407 solution with
stirring. Accurately weighed KP was dissolved in 30 ml
of ethanol and the ethanolic solution of drug was added
slowly in the previously prepared polymer gel with
stirring at 400-600 rpm. Penetration enhancer OA was
added with stirring. The final quantity was made up to
100 g with distilled water. The prepared gel was kept for
24 h at room temperature for complete polymer
desolvation.

Bioadhesive testing
The method developed by Kim et al., 1999 and Choi et
al., 2003 was slightly modified for studying the
bioadhesive character of the prepared gels. The modified
Table 1: Composition of the bioadhesive gel formulations

Batch code
X05
2.5
-
1
-
1
30
5
q.s
Ingredients
S
2.5
1
-
-
1
30
0
q.s
S05
2.5
1
-
-
1
30
5
q.s
S010
2.5
1
-
-
1
30
10
q.s
S015
2.5
1
-
-
1
30
15
q.s
X
2.5
-
1
-
1
30
0
q.s
X010
2.5
-
1
-
1
30
10
q.s
X015
2.5
-
1
-
1
30
15
q.s
P
2.5
-
-
22
1
30
0
q.s
P05
2.5
-
-
22
1
30
5
q.s
P010
2.5
-
-
22
1
30
10
q.s
P015
2.5
-

22
1
30
15
q.s
Ketoprofen (g)
Na CMC (g)
Xanthan gum (g)
Poloxamer 407 (g)
Carbopol 934P (g)
Ethanol (ml)
Oleic acid (ml)
Distilled water (g)*

*Final quantity was adjusted with distilled water to 100 g for constant 2.5%w/w of ketoprofen containing gel.

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S Singh et al.
Pak. J. Pharm. Sci., Vol.22, No.2, April 2009, pp.193-198
195
apparatus used in the study is shown in fig. 1. The
apparatus used for study comprised of two arm balance,
one side of which contains two glass plates and other side
contains a container. One of the two glass plates were
attached permanently with the base of the stage and other
one attached with the arm of the balance by a thick strong
thread. The membrane used for bioadhesive testing was
fresh rat intestinal membrane. Fresh rat intestine was
glued to the upper side of the lower plate and another was
glued to the lower side of the upper plate by using
cyanoacrylate adhesive. The weighed gel (0.5 g) was
placed on the rat intestine glued to the upper side of the
lower plate. Then upper plate was placed over the lower
plate and 50 g preload force (or contact pressure) was
applied for 5 min (preload time). After removal of the
preload force, the water kept in a bottle at some height
was siphoned in the container (fig. 1) at the rate of 10 ml
per min till the plates were detached from each other. The
rate of dropping of water was controlled with on-off
switch same as in infusion bottle. The weight of water
required for detachment of glass plates was considered as
the bioadhesion force of the applied gel.



Fig. 1: Schematic diagram of fabricated modified
bioadhesive force testing apparatus.

Viscosity studies
The viscosities of different KP bioadhesive gels were
determined using a cone and plate geometry viscometer
(Brookfield digital viscometer-III Rheometer V 3.3 HB)
(spindle: 51 cps) at 10 rpm and 25 ± 0.1oC.

In vitro permeation studies
The percutaneous permeation studies were done by using
modified Franz diffusion cell (Diameter of 1.5 cm with a
diffusional area of 1.76 cm2) and membranes used were
shaved, full thickness, excised rat abdominal skin
(Santoyo et al., 1995; Schmook et al., 2001).

Collection and preparation of rat abdominal skin samples:
The animal study protocol was reviewed and approved by
the Animal Ethics Committee at the Department of
Pharmaceutics, Institute of Technology, Banaras Hindu
University, India. Male wistar rats weighing 160-180 g
were used to excise full thickness skin. Rats were
anaesthetized by ether and then hair of abdominal skin
was removed by using electric clipper. Special care was
taken while removing hairs, so as not to destroy the
stratum corneum. A piece of full thickness skin sample
(2.5 x 2.5 cm) was then excised from the shaved
abdominal region. The dermal side of the skin was
cleaned off any adhering subcutaneous tissues and/or
blood vessels and the skins free of any adhering
subcutaneous tissues and/or blood vessels were used for
the permeation study.

In vitro permeation studies of gels: Prepared skin samples
(rat skin) was mounted on the receptor compartment of
the permeation cell with the stratum corneum facing
upward and the dermis side facing downward. The donor
compartment was kept on the receptor compartment and
secured tightly with the help of clamps. The receptor
compartment was then filled with 30 ml of pH 7.4
phosphate buffer. The temperature of media was
maintained at 37 ± 0.5oC with the help of temperature
controlled water jacket. Weighed bioadhesive gel or
marketed gel (2 g equivalent to 50 mg of KP) was then
placed in the donor compartment. The receptor
compartment containing pH 7.4 phosphate buffer was
stirred at 200 ± 5 rpm to maintain the hydrodynamics of
the receptor fluid. Sampling (0.5 ml) was carried out at
the different intervals up to 8 h. The volume of release
media was maintained by adding equal volume of the
fresh media after every sampling. The concentration of
KP in the sample was measured spectrophotometrically at
258 nm.

Anti-inflammatory effect test
The anti-inflammatory activity of KP bioadhesive gels
were evaluated with rat paw edema test. The bioadhesive
gel or marketed gel (1 g) was applied to the shaved
abdominal skin of male Wistar rats weighing 180-200 g.
Just before administration of bioadhesive gels, 1%
carrageenan-saline solution (0.1 ml) was injected into
each hind paw of rats. The thickness of paw edema
induced by carrageenan was measured by using a standard
screw gauge during 8 h after application of KP
bioadhesive gel.


STATISTICAL ANALYSIS

Results are given as mean ± standard deviation (S.D). The
cumulative amount (mg/cm2) of KP permeation through
rat skin was plotted as a function of time (h). The slope of
the linear portion of the plot is presented as the flux
(mg/cm2/h). The results were analyzed statistically using
Student’s t-test or one way analysis of variance
(ANOVA). Significance was determined at P < 0.05.

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Enhanced transdermal delivery of ketoprofen from bioadhesive gels
Pak. J. Pharm. Sci., Vol.22, No.2, April 2009, pp.193-198
196
RESULT AND DISCUSSION

Physicochemical characteristics of ketoprofen gel
The results of bioadhesive force and viscosity of KP
bioadhesive gels were given in table 2.

The concentration of the different polymers NaCMC, XG
and PL407 was selected to obtain the bioadhesive force
between 80-100 gf and viscosity between 700-1000 cps
(table 2). Increasing the concentration (5% v/w to 15%
v/w) of penetration enhancers (OA) did not show any
significant difference (P<0.05) in the bioadhesive force
but significantly decreased (P<0.05) the viscosity of the
bioadhesive gels.

Table 2: Bioadhesive force and viscosity of different
bioadhesive gels.

Bioadhesive
force (gf)
(mean ± S.D*)
S
96 ± 3.0
S05
97± 5.2
S010
95± 7.4
S015
92 ± 1.2
X
85 ± 4.3
X05
86 ± 3.0
X010
84 ± 4.0
X015
80 ± 2.1
P
98 ± 7.6
P05
101 ± 4.8
P010
99 ± 6.9
P015
100 ± 1.5
Marketed gel
88 ± 4.4

* n=5; S.D: standard deviation for five determinations
** n=3; S.D: standard deviation for five determinations

Permeation studies using rat abdominal skin
As expected the flux of KP from OA containing
bioadhesive gels were found significantly higher (P<0.05)
than the flux of KP bioadhesive gel without OA (table 3).
Increasing the concentration (5% v/w to 15% v/w) of
penetration enhancers (OA) showed significant difference
(P<0.05) in the flux of KP (Shin and Cho, 2006). The
highest flux and enhancement factor for KP from the
bioadhesive gels containing OA were found to be 0.421 ±
0.032 mg/cm2/h (in the batch PO15) and 29.71 (in the
batch XO15) respectively.

The highest cumulative amount permeated of KP at 8 h
from bioadhesive gels of NaCMC, XG and PL407 with
OA was found to be 2.220 ± 0.067 mg/cm2, 3.649 ± 0.072
Batch code
Viscosity (cps)
(mean ±
S.D**)
793.4 ± 1.52
785.6 ± 0.89
779.5 ± 2.01
760.1 ± 2.2
789.5 ± 1.05
771.6 ± 1.87
765.3 ± 1.54
760.5 ± 2.54
1174.6 ± 0.25
1092.1± 2.36
1080.5 ± 2.45
1054.1± 1.25
759.1± 0.25
mg/cm2 and 4.119 ± 0.196 mg/cm2, respectively. The
maximum amount permeated from bioadhesive gels of
PL407 can be attributed to the surfactant effect of the
PL407 (Santoyo et al., 1995).

Table 3: Permeation parameters of ketoprofen from
different bioadhesive gels through rat abdominal skin
(mean ± S.D, n=3)

Rat abdominal skin
Batch code
Flux (mg/cm2/h)
Enhancement
factor
1.00
5.63
7.40
10.00
1.00
14.35
22.57
29.71
1.00
1.67
3.05
5.01
-
S
S05
S010
S015
X
X05
X010
X015
P
P05
P010
P015
Marketed gel
0.022 ± 0.009
0.124 ± 0.021
0.163 ± 0.031
0.220 ± 0.029
0.014 ± 0.008
0.201 ± 0.022
0.316 ± 0.034
0.416 ± 0.030
0.084 ± 0.009
0.141 ± 0.021
0.257 ± 0.012
0.421 ± 0.032
0.159 ± 0.014

Batches with higher concentration of OA (batch SO15,
XO15 and PO15) showed maximum fluxes of 0.220 ±
0.029, 0.416 ± 0.030, and 0.421 ± 0.032 mg/cm2/h,
respectively. The flux value for the marketed gel was
found to be 0.159 ± 0.014 mg/cm2/h which was three fold
less than the PO15.

The addition of OA increased the amount of drug
permeated across the rat abdominal skin in all the
bioadhesive gels. The increase in the concentration of
penetration enhancer from 5%v/w to 15%v/w, as
expected, increased the amount permeated and the flux
(table 3 and fig. 2).

Anti-inflammatory study of selected bioadhesive gels
The in vivo pharmacodynamic studies were carried out by
rat paw edema test on three selected bioadhsive gel
formulations (batch SO15, XO15 and PO15) and the
inhibition of swelling was compared with controls
(without penetration enhancer) and the marketed gel. The
gels without penetration enhancer (OA) showed less
inhibition of the swelling or inflammation (fig 3). A
significant inhibition (P<0.05) of inflammation was found
with the gels containing OA in comparison to the gels
without OA. There was no significant (P>0.05) difference
in the inhibition of inflammation in between the gel SO15
and XO15. However inhibition of inflammation shown by

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Pak. J. Pharm. Sci., Vol.22, No.2, April 2009, pp.193-198
197
the gel PO15 was significantly greater (P<0.05) than the
both SO15 and XO15. The inhibition of inflammation was
studied for the marketed gel and compared with the
prepared gels containing OA. It was found that the
inhibition of inflammation by the marketed gel was
significantly less (P<0.05) than the all three selected
bioadhsive gel formulations. The in vivo results were
found to be in correlation with the in vitro permeation
results.

0
0.5
1
1.5
2
2.5
0 0.512345678
Time (h)
Cumulative amount permeated (mg/cm2)
S
SO5
SO10
SO15

(a)

0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.512345678
Time (h)
Cumulative amount permeated (mg/cm2)
X
XO5
XO10
XO15

(b)

0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
012345678
Time (h)
Cumulative amount permeated (mg/cm2)
P
PO5
PO10
PO15
Marketed gel


(c)

Fig. 2: Cumulative amount of ketoprofen permeated from
the gels containing sodium carboxymethylcellulose (a),
xanthan gum (b), poloxamers 407 (c) through the rat
abdominal skin

0
5
10
15
20
25
30
35
40
45
50
00.512345678
Time (h)
Percent swelling
S
SO15
X
XO15
P
PO15
Marketed Gel


Fig. 3: Percentage swelling of carrageenan induced rat
paw inflammation showing anti-inflammatory effect of
ketoprofen bioadhesive gels and marketed gel.

CONCLUSIONS

This study demonstrated that incorporating KP into
bioadhesive gels containing a penetration enhancer in
various concentrations enhanced the drug permeation
through rat skin and in vivo performance (table 3 and figs.
2-3). Bioadhesive gels of KP containing OA may offer
promise as an anti-inflammatory dosage form, ensuring
more effective therapy, but additional experiments should
be performed before the formulation is used in humans.

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