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Journal of Research (Science), Bahauddin Zakariya University, Multan, Pakistan.
Vol. 17, No. 3, July 2006, pp. 165-171 ISSN 1021-1012
Syed Nisar Hussain Shah*, Mahboob Rabbani
and Muhammad Fakhruddin Amir
Faculty of Pharmacy, Bahaudin Zakariya University, Multan-Pakistan. Abstract
In the present study, the effect of Urea as an enhancer on transdermal
absorption of 1% Diclofenac Diethylamine (Non-steroidal Anti-
inflammatory Drug) through hairless rabbit skin was evaluated in-vitro
study at various concentrations to improve the skin permeability.
From the data, Urea shows slightly large lag time gives a picture about
its enhancing effect. The permeability rate constant is almost
approaching to 1 in almost all the concentrations of urea. The
permeability co-efficient of Diclofenac Diethylamine did not change
under the influence of urea at concentration 0.1, 0.2 and 0.3% but it was
increased by increasing the concentration of urea.
Keywords: Diclofenac, enhancer, topically, transdermal.
In the severe renal failure Urea can create precipitates on the skin surface. It is also possible to find increased urea concentration in the skin layers. Urea is a physiological component of NMF (natural moisturizing factor) of the skin. Owing to its mild keratolytic effect, urea is used in the topical drug formulations. Urea and its analogues were also studied as possible candidates to accelerate percutaneous absorption of drugs [Wong et al.1988, Williams and Barry 1989]. Transdermal drug delivery could provide constant drug release for days, avoids first-pass metabolism, and could allow drug effects to be rapidly terminated by simply removing the patch. However, absorption across the skin for molecules larger than 1000 Daltons has proved to be difficult, even with the addition of permeation enhancers [Rodney and Gibaldi 2003]. The amount of drug bioavailable for targeting the sites of action is lower than via oral route, but the absorbed dose appears to be adequate for therapeutic use [Katz and Poulsen 1971]. Many Non-steroidal anti-inflammatory drugs (NSAIDs), are among the most commonly prescribed drugs worldwide and are responsible for approximately quarter of all adverse drug reports [Roberts II and Morrow 2001]. They are used in the treatment of osteo- and rheumatoid arthritis as well as local inflammation. [Roberts 1999, Hadgraft et al. 2000]. The well-known side effects observed after oral administration of NSAIDs have accelerated the development of alternative 165 J. res. Sci., 2006, 17(3), 165-171
Syed Nisar Hussain Shah, Mahboob Rabbani and Muhammad Fakhruddin Amir pharmaceutical forms such as topical cream, gel, lotion, foam and Transdermal-patch formulations that allow local absorption at the inflammation site without adverse systemic reactions [Rhee et al. 2001, Djordjevic 2003]. MATERIALS AND METHODS
Diclofenac Diethylamine supplied by Novartis (China origin), the enhancer used
was Urea (Merck). Double distilled water from an electrically heated still, having
the pH 6.8 ± determined by the pH meter and stored in a well leached amber
glass bottle, was used throughout the experiment. Ethanol (Merck), sodium
acetate (Merck) and sodium chloride (Merck) were used. HPLC was used for the
analysis of the sample taken using column C18, 5 µm, 150 mm length, 4.5mm
internal diameter (Neocleocil; Alltech) and pump WATER’S 600E, Detector
WATER’S 484, using the HPLC software Millennium Version-2.15 (courtesy
Novartis Pakistan).
In-vitro technique that was used to study transdermal absorption involves the use
of animal excised skin; in many cases full thickness is used [Hadgraft et al.

An isocratic mobile phase was used containing 0.1 M sodium acetate.
1 gram of Diclofenac Diethylamine was dissolved in 5 ml methanol in 100 ml
volumetric flask and the volume was made up to the mark with normal saline.
This was used as reference control solution without any enhancers.
Test solutions were prepared by dissolving 1 gram of Diclofenac Diethylamine in
5 ml methanol in 100 ml volumetric flasks and the solutions were made up to the
mark with previously constituted solutions of 0.5 and 1% enhancer (Urea) in
normal saline.
Diffusion cell was fabricated after Franz [1975], Keshary [1984], Tojo [1985],
Cordero et al. [1996], Takahashi [1996], Valenta [2000] with some modifications.
The cell was in the form of two cylindrical glass half cells. The inside diameter
was 2 cm. The diffusion cell halves were termed as upper half cell (donor
compartment) and the lower half cell (receptor compartment). The volume
capacity of the donor and receptor compartments was 40ml and 35ml,
respectively. The membrane was mounted in between the two half cells and the
exposed penetration area was approximately 3.14cm2. From the lower half of the
receptor compartment at a distance of about 3.8 cm a side arm 4 cm in length is
used for taking the sample and correcting the volume of receptor compartment
with the help of saline solution by exposing the epidermal side toward the donor
EFFECT OF UREA ON TOPICAL ABSORPTION OF DICLOFENAC DIETHYLAMINE … 167 half cell. The two half cells after clamping was mounted on a magnetic stirrer and
small magnetic fleas were placed in the receptor compartment and the receptor
solution is stirred at 60 rpm.


The membrane, full thickness skin was taken from the abdominal surface of the
hairless rabbit. The skin at the lower abdomen was marked and was shaved and
then rabbit skin was sacrificed and whole skin was removed and a rectangular
section marked was excised from the animal with surgical scissors. Since the
skin was not firmly attached to the viscera it was lifted easily from the animal
after the incision was made. Prior to the skin removal, a uniform circle was made
on the abdomen, marking the precise skin section to be positioned between the
two half cells after the excised skin was trimmed into an oversized rough circle it
was mounted between the half cells with the marked section centered. The skin
was placed in a normal saline solution before mounting on to the diffusion cell
[Durrheim et al. 1980, Cordero et al. 1996].
Skin was cut according to the diameter of the diffusion cell and the half cell was
held fast by a clamp stretching of the skin as evidenced by distortion or
expansion of the circular outline was corrected and the half cell were held fast by
a clamp.
The receptor cell filled with normal saline was stirred by magnetic stirrer at 60
rpm for 30 minutes, at which time the compartments were evacuated with a
syringe and refilled with fresh normal saline. Then the compartments were
evacuated a second time refilled evacuated a third time, and finally refilled with
normal saline. The donor compartment of the cell was charged with a test


The donor compartment of the cell was charged with a test solution containing
1% of Diclofenac Diethylamine plus different concentrations of enhancer, which
was dissolved in 100 ml of normal saline. The receptor cell contents were stirred
and at predetermined times, sample were taken and transferred to the small
bottles having stoppers, using 10 ml syringe the time of charging the donor
compartment was noted at the beginning of the diffusion runs and the receptor
samples were reference to this time.
From the side arm of the receptor compartment, 5 ml of the sample was drawn
each time at an interval with the help of 5 ml syringe and correcting the receptor
half cell volume with normal saline. The 5 ml sample is drawn at an interval of 5
minutes for 30 minutes.
From the sample taken from the receptor cell, a portion of 10 micro-liters was
taken and was run on the HPLC having a column C18 (Neocleocil, Alltech),
pump (WATER’S 600 E), detector (WATER’S 484) using the HPLC software
Millennium Version-2.15 at a flow rate of 0.8 ml per minute, by using auto
sampler at the wavelength of 254 nm.
Syed Nisar Hussain Shah, Mahboob Rabbani and Muhammad Fakhruddin Amir PHARMACOKINETIC ANALYSIS OF IN-VITRO TRANSDERMAL

Data of the in-vitro transdermal absorption study using stripped skin were
analyzed on the basis of the Pharmacokinetic model shown in Fig. 1.
K12 K23
Fig. 1: Pharmacokinetic model for the analysis of in-vitro transdermal absorption study.
The permeation profile of the receptor phase concentration in microgram per
100ml is summarized in Table 1. The lag Time of the plots was calculated
graphically by extrapolation from the pseudo-steady-state region of the graph of
the total amount penetrated versus time to the X-axis. The Diffusion Coefficients
and Permeability Coefficients of different concentrations of enhancer were
calculated by dividing the square of the thickness of the rabbit incised skin by 6 x
Lag Time [Badar 1992]:
It was calculated using the following relation:
where h is the thickness of the rabbit skin and L is the Lag Time.
It was calculated by dividing the Diffusion Coefficient by square of the effective
absorption area of the skin in contact [Tsai et al. 2001]:
where A is the effective absorption area of the skin in contact.
As a measure of the penetration enhancing activity of Urea, the enhancement
ratio (ER) was calculated as under [Abdullah et al. 1996]:
ER = P after application of penetration enhancer P before application of penetration enhancer EFFECT OF UREA ON TOPICAL ABSORPTION OF DICLOFENAC DIETHYLAMINE … 169 Typical results have been shown in Table 1. The data will be subjected to proper
relevant statistical analysis.
Table 1: Effect of different concentrations of Urea on the permeability parameters of l% Diclofenac
Diethylamine using hairless rabbit skin. When Value of Permeation co-efficient (P) for sample without enhancer was 0.000173.
The Flux of a drug is directly proportional to its thermodynamic activity:
Flux, which measures the mass of material transported through the skin, is more
relevant parameter, therapeutically, than the Permeability Coefficient [Rautio
These values indicate that the penetration may be dependent on the lipoidal
solubility of the drug moiety. However, the permeation may be complicated by
charge effect and also may depend on the skin Partition Coefficient of the drug
between the aqueous phase and lipid phase of the barrier [Shah et al. 2005].
The Partition Coefficient (Pc) can be calculated using the expression:

The Diffusion Coefficients presented in Table 1 reflects its effects on Permeability
Coefficients of Diclofenac Diethylamine. The change in lag Time, changes the
Diffusion Coefficients of Diclofenac Diethylamine that increases with decrease in
Lag Time [Aguiar and Weiner 1969 and Durrheim et al. 1980].
Finally, the permeability rate constant of various concentrations of urea was
calculated which are also summarized in the same Table 1. As was assumed
that the whole penetration process is first order rate constant, the rate constant
then can be calculated as under [Badar 1992]:
Rate Constant = Log (y2 - y1) × 2.303
Syed Nisar Hussain Shah, Mahboob Rabbani and Muhammad Fakhruddin Amir CONCLUSIONS
Urea shows fluctuating behaviour as the permeability rate constant is almost approaching to 1 in almost all the concentrations of urea. The permeability co-efficient of Diclofenac Diethylamine did not change under the influence of urea at concentration 0.1, 0.2 and 0.3% but it was increased by increasing the concentration of urea. From the data, the following points can be concluded: 1. In the interpretation of results the lag time plays an important role. Urea shows the larger lag time gives a picture about its slow enhancing effect as compare to samples without Urea. 2. The permeability coefficient calculated for Diclofenac Diethylamine under the influence of Urea shows enhancing characteristics. 3. The flux rate of Diclofenac Diethylamine in the presence of Urea shows that the penetration of drug through hairless rabbit skin almost increased. 4. The Diffusion Co-efficient of the drug increases all the way up to 1.0% of urea showing the steady increase initially and a sharp one in the end.
The researchers greatly acknowledge the co-operation extended by Novartis
Pakistan Ltd., Jamshoro, Hyderabad and also Bahauddin Zakariya University for
providing financial support for this research project.


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