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Journal of Food and Drug Analysis, Vol. 20, No. 2, 2012, Pages 464-470

doi:10.6227/jfda.2012200205

Developing an Efficacious Diclofenac Diethylamine

Transdermal Formulation
SYED NISAR HUSSAIN SHAH1, MEHBOOB-E-RABBANI1, YASSER SHAHZAD2,
AMIR BADSHAH3, VICTOR M. MEIDAN4 AND GHULAM MURTAZA5*
1.

Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan

2.
School of Pharmacy, University of Huddersfield, UK
3.

Department of Pharmacy, University of Peshawar, Peshawar, Pakistan

4.

5.

SIPBS, University of Strathclyde, Glasgow, Scotland, UK

Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan
(Received: November 8, 2011; Accepted: January 31, 2012)

ABSTRACT
The aim of current study was to develop an efficacious topical formulation of diclofenac diethylamine (DDA), for eventual use as a
locally applied analgesic lotion. A series of lotions were prepared that incorporated 2% w/v DDA but a variable (1, 2, 3 or 4% v/v) concentration of oleic acid (permeation enhancer)-turpentine oil binary mixture (1:1 v/v). After measurements of each lotions visual appearance,
viscosity, spreadability, pH and long term stability, a diffusion cell method was employed to quantify DDA permeation from each lotion
across both silicone membranes and rabbit skin. Subsequently, three distinct in vivo animal models were utilised to evaluate each lotions
induced anti-inflammatory, antinociception and irritancy effects. Lastly, a panel of human volunteers assessed the subjective sensory feel of
each lotion as well as its potential for cutaneous irritancy following a brief application. Upon preparation, all four DDA-containing lotions
(L1, L2, L3 and L4) appeared as clear, colourless, homogenous and aggregate-free solutions. All the lotions exhibited a pH of ~6.2 with
non-significant (p > 0.05) differences between each formulation (data not shown). Increasing the formulations oleic acid concentration
yielded a general trend of mild enhancement of DDA transport across silicone membrane and dramatic improvements of DDA flux through
rabbit skin. Application of each of the DDA-containing formulations significantly reduced tissue inflammation in the rabbit model. It was
determined that the DDA lotion containing 4% v/v oleic acid exhibited the best performance overall and that this specific formulation
should be the basis for further clinical investigation.
Key words: Diclofenac diethylamine, oleic acid, transdermal, percutaneous absorption, lotion.

INTRODUCTION
Diclofenac is an established nonsteroidal anti-inflammatory drug (NSAID) that is widely used to symptomatically
alleviate the pain and swelling associated with conditions
such as arthritis, toothache, dysmenorhea and other musculoskeletal disorders(1). Unfortunately, oral diclofenac use is
associated with extensive first pass metabolism as well as
potentially gastrointestinal irritation. Intramuscular injection
of the drug can cause skin lesion formation. Consequently,
the transdermal delivery of diclofenac has attracted considerable interest in recent years(2,3). In particular, research has
tended to focus on developing effective topical formulations
of diclofenac diethylamine (DDA)(2,3), which is the diethylammonium salt of this NSAID. In terms of physicochemical
* Author for correspondence. Mobile: 00923142082826;
Fax: 0092992383441; Email: gmdogar356@gmail.com

properties, DDA exhibits a molecular weight of 369 Da, a

melting point of 157C and a log D (pH 7.4) of 0.85(4). Pharmacologically, DDA has a bioavailability of 40-60%, a short
biological half life of 2-3 h and small dose requirements of
25-50 mg(5). Taken together, all these characteristics suggest
that DDA should be a good candidate for transdermal dosage
form development(6).
Over the last decade, researchers have explored
different techniques for maximizing DDA delivery across
skin. Published approaches have included incorporating the
drug into emulsion-based systems(7-9), entrapment within
niosomes(10), use of different vehicle-chemical enhancer
combinations(7,11-14) and encapsulating the drug within
lyotropic liquid crystals(15) or discoid-shaped bilayer aggregates termed bicelles(16).
Chemical substances temporarily diminishing the
barrier of the skin and known as accelerants or sorption

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Journal of Food and Drug Analysis, Vol. 20, No. 2, 2012

promoters can enhance drug flux. Several types are known

as (i) sulphoxides and similar chemicals, (ii) azone, (iii)
pyrrolidones, (iv) fatty acids, (v) essential oil, terpenes and
terpenoids, (vi) oxazolidinones and (vii) urea. Oleic acid is a
fatty acid and is found in excess in nature especially in human
skin. Its capability as a skin penetration enhancer has been
normally recognized in a variety of in vitro studies(17).
No previous study has elaborated the formulation of
DDA as lotion. In this presentation, a series of topical lotions
has been prepared all containing a fixed concentration of
2% v/v DDA but a variable concentration of an oleic acidturpentine oil (a permeation enhancer) binary mixture to
provide penetration enhancement. Initially, measurements
were conducted to determine pH, viscosity, spreadability and
drug content of each lotion. Then, Franz cells were used to
quantify DDA permeation from each lotion across silicone
membranes and rabbit skin. Some of these in vitro studies
were repeated within the context of accelerated stability
testing. In subsequent in vivo animal studies, induced antiinflammatory, anti-nociception and irritancy effects of each
lotion were assessed by the carrageenan rat paw edema
assay, the hot water-tail flick test and Draize skin irritation
methods, respectively. Finally, lotions were briefly assessed
by human volunteers for their subjective sensatory effects as
well as potential for cutaneous irritancy. The overall aim was
to determine which oleic acid concentration should be incorporated into the formulation in order to produce the most
efficacious transdermal DDA lotion.

MATERIALS AND METHODS

I. Materials
Oleic acid, methanol and sodium acetate were purchased
from Merck (Darmstadt, Germany). Isopropyl alcohol and
gamma carrageenan No. 2249 were from Fluka (Buchs, Switzerland). Carbomer 980 was from Fisher Scientific (Schwerte,
Germany). Turpentine oil was from MS Traders (Guangdong,
China). Reference standard sample of DDA (99.8%) powder
was a gift from Novartis (Jamshoroo, Pakistan). Silicone
membranes of 400 m-thickness were purchased from Samco
(San Fernando, USA) while silicone grease was purchased
from Dow Corning (Midland, USA). Normal saline was
prepared by dissolving sodium chloride (Merck, Darmstadt,
Germany) in double distilled water at 0.9% w/v strength.

(LC) was prepared by dissolving 2 g DDA in 15 mL ethanol

and adding normal saline to make a 100 mL volume.
III. In Vitro Characterization Studies
Each DDA-containing lotion was subjected to test its
pH, viscosity, spreadability, homogenity and drug solubility.
Each test was conducted in triplicate (n = 3).
The pH of lotion was measured on a digital pH meter
(Mettler & Toledo, Giessen, Germany). Viscosity were
measured at room temperature (25 2C) on a Model RVTDV
II Brookfield viscometer (Stoughton, USA). A C-50 spindle
was employed with a rotation rate of 220 rpm. The gap value
was set to 0.3 mm.
The spreadability of each lotion was determined by
the wooden block and glass slide method as detailed previously(18). Essentially, 5 mL (100 mg) of lotion was added to
a dedicated pan and the time taken for a movable upper slide
to separate completely from the fixed slide was noted. The
spreadability was determined according to the formula:
S = M.L/t
Where
S = Spreadability
M = Weight/Volumes tide to upper slide
L = Length of glass slide
t = Time taken to separate the slides completely from
each other
Each formulated lotion was evaluated for homogeneity by naked eye examination. This involved a subjective
assessment of appearance including the presence of any
aggregates(19).
Each DDA-containing lotion was also assayed for drug
content. Five milliliter (100 mg) aliquot of the test lotion
was dissolved in 5 mL of ethanol and then PBS (pH 7.4) was
added to make up to 100 mL. The mixture was rocked for 2 h
on a mechanical shaker before being filtered. The supernatant
was then assayed for DDA by HPLC.
IV. Accelerated Stability Studies
All the formulated lotions were subjected to a 3 monthlong protocol of accelerated stability testing conducted at a
temperature of 40 2C, 75% RH. At 12 h, 1 day, 7 days,
1 month and 3 months, each formulation was examined
for changes in appearance, pH, viscosity and drug content.
Again, these experiments were performed in triplicate (n = 3).

II. Preparation of Diclofenac Lotions

V. HPLC Assay

Preparation of all the enhancer-containing diclofenac

lotions (L1, L2, L3 and L4) involved initially dissolving 2 g
of DDA in 15 mL ethanol. Then selected volume (1, 2, 3 or
4 mL) of oleic acid : turpentine oil (1 : 1 v/v) mixture was
added. In all cases, 1.5 mg carbomer 980, 20 mL phosphate
buffered saline (PBS, pH 7.4) and 1 mL isopropyl alcohol
were added into the mixture. Lastly, ethanol was added to
make up a 100 mL volume. An enhancer-free control solution

Quantitative analysis of DDA was performed on a

Waters HPLC system (Elstree, UK) equipped with a 600E
pump, a 484 UV-visible detector, an autosampler and a C18
Nucleosil 5 m column of 150 mm length and 4.5 mm
internal diameter (Alltech Associates, Deerfield, USA).
A dedicated HPLC software package called Millenium
(Ver. 2.15, Novartis, Jamshoroo, Pakistan) was utilized for
output data analysis. An isocratic mobile phase consisting of

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Journal of Food and Drug Analysis, Vol. 20, No. 2, 2012

methanol : 0.1 M sodium acetate mixture (70:30; v/v) was

filtered and degassed by sonication. The elution parameters
were a flow rate of 0.8 mL/min and an injection volume of
10 L. The detection wavelength was 276 nm and the DDA
retention time was 5.5 min. An internal standard of propylparaben showed a retention time of 4.1 min. Intra- and interday RSD for DDA was below 4% while intra- and inter-day
accuracy was greater than 95%; the assay methodology met
the validation guidelines of the International Conference of
Harmonization (ICH), i.e. calibration curve was linear in a
concentration range of 10-60 g/mL with a regression coefficient (R2) of 0.9791. The percentage recovery of DDA was
98.95%. The limit of detection was 0.225 g/mL while the
limit of quantitation was 0.350 g/mL.
VI. Permeation Studies
Full thickness skin was excised from the abdomens of
male White New Zealand rabbits (3 to 4 kg) that had the hairs
clipped short. After excision, it was necessary to carefully
remove adhering subcutaneous fat. Then, the skin was cut
into samples that were just larger than the surface area of the
diffusion cells. Prior to mounting in the cells, the skin samples
were briefly immersed in normal saline(19,20). For the silicone
membrane studies, pieces of size suitable for mounting in
Franz cells were cut out and then soaked overnight in PBS
(pH 7.4). This procedure was performed to remove excipients present within the membrane upon purchase(21).
Permeation measurements were made using Franz cells
manufactured in house, exhibiting a diffusion area of 0.85
cm2 and a receptor cell volume of 4.5 mL. Each receptor
compartment, containing a small magnetic stirring rod,
was initially filled with ultrasonically-degassed PBS (pH
7.4). Subsequently, the test membrane (either rabbit skin or
silicone) was inserted as a barrier between the donor and
receiver cells. Silicone grease was applied to create a good
seal between the barrier and the two Franz compartments.
To start each permeation experiment, 1 mL volume of each
lotion formulation was deposited in the donor cell, which was
then covered with parafilm. Uniform receptor phase mixing
was initiated by placing the diffusion cells on a magnetic stirring bed immersed in a water bath (35 2C). After 1 h, the
receptor solution was removed and completely replaced with
fresh, pre-warmed PBS. At scheduled times, an aliquot of
0.5 mL receiver fluid was withdrawn and the receiver phase
was replenished with 0.5 mL fresh PBS. Withdrawn aliquots
were assayed by HPLC but with the derived DDA concentration values being mathematically corrected for the progressive dilutions caused by repeated sampling. Sink conditions
existed throughout the experiment. Since skin exhibits large
sample-to-sample permeability differences(19), each experiment consisted of 5 replicate runs (n = 5).
VII. In Vivo Animal Studies
In vivo study consisted of three separate types of studies.
All were conducted under conditions that had been regulated

and approved by the Animals Ethics Committee of Bahauddin

Zakariya University, Multan (Pakistan).
Each DDA-containing formulation was evaluated for
its anti-inflammatory potency by means of the carrageenaninduced rat paw edema assay(22). The assay was run on male
Wistar rats (150 5 g) purchased from the Institute of Biotechnology of Bahauddin Zakariya University, Multan (Pakistan).
These rats were allowed free access to food and water. The
protocol involved injecting 0.1 mL of 1% w/v carrageenan
suspension in normal saline into the sub-plantar tissue of each
animals right hind paw. This was immediately followed by
applying 1 mL of the DDA-containing lotion over a 2 cm2
area of injection site. After 3 h, the extent of tissue inflammation was quantified by measuring the linear paw circumference(23). Each test lotion was applied to 3 rabbits.
In the next set of in vivo studies, each analgesiccontaining lotion was evaluated for its antinociception effect
by running a modified version of the established hot watertail flick test(24) on male Wistar rats ( 450 g weight). To this
end, 1 mL aliquot of test formulation was applied to each
animals abdomen. The animal was placed in a dedicated
cloth restrainer that was specially designed for this version
of the flick test(25). At 30, 45 and 60 min after lotion administration, animals tail (2-5 cm long) was immersed in water
maintained at 53 1C. The reaction time was the time
taken by the rat to flick its tail. In practice, first reading was
ignored and reaction time was taken as the mean of subsequent two readings. Each analgesic formulation was tested
on 3 different rats.
Lastly, each formulation was assessed for irritancy by
conducting modified Draize skin irritation tests(26) on male
White New Zealand rabbits (3-4 kg) obtained from Aventis
Pharmaceuticals (Jamshoroo, Pakistan). For this purpose, a
dorsal area on each restrained animal was shaved and then
tape-stripped three times to detach several upper layers of
the stratum corneum. Point five milliliter of each test lotion
was applied to these areas, which were then covered with a
plastic patch. After 4 h, patch was removed and rabbits were
observed over 14 days for signs of erythema, edema and
ulceration. On days 1, 3, 7 and 14, visually apparent cutaneous changes were assigned scores ranging between 0 and
4 with higher numbers signifying greater skin damage. Each
DDA formulation was tested on 3 rabbits.
VIII. Clinical Studies
The double blind clinical trials involved eleven Caucasian volunteers, both male and female, ranging between 20
to 24 years in age. A small amount of test formulation was
applied to a 12 cm2 area on the back of each volunteers
hand and left on for 10 min. Each volunteer rated the test
lotions effects in terms of five different subjective sensatory categories. The categories were ease of application,
skin sensation immediately after application, long-term skin
sensation, skin shine (i.e. visual appearance) and perception
of induced skin softness. The rating scale used consisted of
nine integer values ranging between -4 to +4, indicating very

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Journal of Food and Drug Analysis, Vol. 20, No. 2, 2012

After preparation of the DDA-containing lotions, the

methodology consisted of initial characterization studies
including accelerated stability studies, in vitro permeation
studies across synthetic membranes and rabbit skin, various
in vivo animal studies and finally some human volunteer
investigations. In all cases, analysis of the data was carried
by applying a one-way ANOVA with a probability of p < 0.05
set as statistically significant.

RESULTS AND DISCUSSION

I. In Vitro Characterization Data
Upon preparation, all four DDA-containing lotions (L1,
L2, L3 and L4) appeared as clear, colourless, homogenous and
aggregate-free solutions. All the lotions exhibited a pH of
~6.2 with no significant (p > 0.05) differences between each
formulation (data not shown). However, there were variations
in lotion viscosities with L1, L2, L3 and L4 showing respective
mean standard deviation values of (97 0.77) 10-4, (93
0.67) 10-4, (96 0.67) 10-4 and (103 0.78) 10-4
dyn/s/cm-2. With the exception of L1 and L4, each lotions
viscosity was significantly different from that of the others.
With respect to spreadability, L1, L2, L3 and L4 exhibited
respective mean standard deviation values of 4.69 0.13,
3.7 0.13, 2.48 0.03 and 2.08 0.09 mg/cm/s-1. Statistical analysis indicated that these values were significantly
different from each other. Moreover, there is obviously a
clear trend of decreasing spreadability with increasing oleic
acid turpentine oil content.
During the three months accelerated stability testing,
none of the formulations showed any visually apparent
changes in appearance, opaqueness or color. Furthermore,
none of the formulations underwent any statistically significant (p > 0.05) changes in pH, viscosity, spreadability, drug
content or transdermal permeability through rabbit skin over
the three month period.
II. In Vitro Permeation Data
Figure 1 shows the plots of cumulative DDA permeation across silicone membranes as a function of time. It is
noteworthy that the plots exhibited a non-classic profile with
the flux rates initially higher and then actually undergoing
a decrease at ~60 mins. This effect could be due to the silicone membrane material undergoing perturbations, such as
perhaps adsorption of excipients, upon initial exposure to the

Cunulative amount of drug

permeated (g/cm2)

IX. Statistics

donor solution(12). It was decided to select the period 15 to

180 min to calculate the mean flux rates. By this approach, it
was determined that the control lotion (Lc) permeated across
the barrier at a mean rate of 0.038 g/cm2/min with a standard deviation of 0.006 g/cm2/min. In contrast, L1, L2, L3
and L4 exhibited respective mean standard deviation flux
values of 0.066 0.002, 0.095 0.003, 0.095 0.003 and
0.112 0.005 g/cm2/min. The enhancement ratio (ER) is
given by the ratio of the oleic acid-containing lotions mean
flux to the control lotions mean flux. For L1, L2, L3 and
L4, the respective enhancement ratios were 1.75, 2.51, 2.52
and 2.97. All these flux values differed significantly (p <
0.05) from each other except the flux values of L2 and L3 that
were not significantly (p > 0.05) different. Thus, increasing
the formulations oleic acid concentration yielded a general
trend of mild enhancement of DDA transport across silicone
membrane.
Figure 2 presents a graph of cumulative DDA permeation
across rabbit skin as a function of time. One point of interest
is that drug flux more or less ceased beyond ~720 min (12 h).
This is explainable by precipitation that was observed in the
donor cell at this time. This precipitate coated the rabbit skin
thus markedly reducing the surface area available for drug
diffusion(9). Another possible reason of retarded permeation
via rabbit skin could be due to the saturation of drug deposition
within the skin reservoir(29). Consequently, the time period
after 720 min was ignored for calculating flux values. It was
found that Lc delivered a mean transdermal drug permeation
rate of 0.11 g/cm2/min with a standard deviation of 0.0005

40
30
20
10
0

50 100 150 200

Time (min)

L1

L2

L3

L4

Lc

Figure 1. Cumulative permeation of DDA across silicone membrane as

a function of time. Error bars represent SD values, with n = 5.

Cunulative amount of drug

permeated (g/cm2)

bad to excellent respectively. In addition, skin treatment sites

were visually examined for the signs of cutaneous irritancy.
The clinical studies in human were approved by the Departmental Ethical Committee and were conducted according to
the guidelines of Good Clinical Practice(27) and Helsinkis
Declaration regarding human use in experiments(28).

6000
5000
4000
3000
2000
1000
0
0

500 1000 1500

Time (min)

L1

L2

L3

L4

Lc

Figure 2. Cumulative permeation of DDA across rabbit skin as a function of time. Error bars represent SD values, with n = 5.

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Journal of Food and Drug Analysis, Vol. 20, No. 2, 2012

g/cm2/min. In contrast, L1, L2, L3 and L4 exhibited respective mean standard deviation flux values of 4.80 0.031,
5.73 0.086, 5.89 0.003 and 6.04 0.003 g/cm2/min.
These translate to ER values for L1, L2, L3 and L4 of 45.6,
54.5, 56.0 and 57.5, respectively. Notably, these flux values
differed significantly from each other. So augmenting oleic
acid concentration in the topical lotion resulted in dramatic
improvements of DDA flux through rabbit skin.
From above described results, it can be elaborated that
the rate of drug permeation was faster with higher ER values
through rabbit skin as compared to that of silicone membrane.
This difference, of course, can be attributed to the structural
difference of two membranes(3,6,19).
III. In Vivo Animal Data
Table 1 shows the data from the carrageenan challenge
anti-inflammatory tests. It can be seen that application of each
of the DDA-containing formulations significantly (p < 0.05)
reduced tissue inflammation in the rabbit model. In contrast,
application of Lc non-significantly (p > 0.05) affected inflammation. Another noteworthy point is that while L1 was significantly (p < 0.05) less anti-inflammatory than L2, L3 and L4,
the latter three formulations did not differ significantly from
each other in anti-inflammatory potency.
The data derived from the hot tail antinociception
studies are presented in Table 2. The graphs clearly indicated
that the reaction time measured following treatment with a

Table 1. In vivo edema reduction induced by each DDA formulation

in carrageenan-challenged rabbits (Mean SD, n = 3)
Anti-inflammatory activity

Formulated
Lotion

Change in Paw Edema (mm)

SD (n = 3)

L1

0.30.04*

L2

0.50.19*

L3

0.40.24*

L4

0.60.17*

LC
0.10.004**
Note: * represents significant changes, while ** represents non-significant changes.
Table 2. In vivo tail flick response times (antinociception) associated
with each DDA formulation at 30, 45 and 60 min after lotion application (Mean SD, n = 3)
Formulated
Lotion

Hot tail flick test

Time after application of lotion
30 min

45 min

60 min

L1

3.69 1.94

4.21 0.71

4.45 1.03

L2

6.96 0.92

9.07 1.45

10.1 1.81

L3

7.89 0.82

11.3 1.02

13.1 1.14

L4

8.24 1.22

LC

1.39 0.76

12.14 2.1
1.75 0.87

13.69 2.2
2.19 0.93

DDA-containing lotion was always significantly longer than

that with the control lotion (Lc). Furthermore, the extent of
induced antinociception followed the trend L4 > L3 > L2 > L1,
indicating that oleic acid content influenced antinociception
potency.
The results of Draize irritation tests indicated that
application of both LC and L4 was invariably associated
with no skin irritation throughout the entire 14 day period.
With respect to the L1 and L3 formulations, all tested rabbits
showed some mild erythema (score of 1) by day 14 although
not at the earlier observation time. Topical administration of
L2 to all three tested animals yielded mild erythema (score
of 1) on day 7 that subsequently resolved by day 14. The
erythema could be due to the use of oleic acid in formulation
as Boelsma et al.(30) has reported same problem on topical
use of oleic acid.
IV. Clinical Studies Data
The volunteers rated all the DDA formulations as scoring
between 3 to 4 in terms of all categories: ease of application,
skin sensation immediately after application, long-term skin
sensation, skin shine and induced skin softness. No lotion
caused any observable cutaneous irritation.
As far as we could ascertain there are no literature report
documenting the permeation of DDA across either rabbit skin
or silicone membranes. Hence, it was not possible to compare
our flux data with values published by others. Our principal
finding was that oleic acid only moderately enhanced DDA
flux across silicone membrane (ER 2.97) and this effect
was explainable by the presence of oleic acid changing
the drugs thermodynamic activity and/or the vehicle to
membrane partition coefficient. However, inclusion of oleic
acid caused a much greater enhancement of DDA flux across
skin (ER 57.5). This effect results from the fact that oleic
acid promotes phase separation within the intercellular lipids
of the stratum corneum. In particular, the enhancer creates a
more drug-permeable fluid phase in these lipids that co-exists
with the regular crystalline phase of these lipids(31).

CONCLUSIONS
It is noteworthy that the L4 lotion exhibited the most
effective flux enhancement properties. Moreover, in vivo
studies showed that L4 yielded the most potent antinociception activity while not causing any more skin irritation
than the control lotion. It was not less effective than any of
the other DDA formulations in terms of anti-inflammatory
activity in rabbits. Like all the other DDA lotions, L4 showed
good spreadability, viscosity and was found very acceptable by human volunteers. They reported no skin irritation
following brief application of L4. It can be concluded that
this formulation containing 4% v/v oleic acid was the most
optimal overall. Future work will focus on running more
elaborate clinical studies with this particular formulation.

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Journal of Food and Drug Analysis, Vol. 20, No. 2, 2012

DECLARATION OF INTEREST
The authors report absolutely no conflict of interest.

ACKNOWLEDGMENTS
The authors would like to thank Bahauddin Zakariya
University, Multan (Pakistan) for providing financial support
for this research. We also acknowledge the HEC (Pakistan) for
awarding an IRSIP scholarship to the corresponding author,
as this allowed him to work on this project at the School of
Pharmacy (London, UK).

REFERENCES
1. Gan, T. J. 2010. Diclofenac: an update on its mechanism
of action and safety profile. Curr. Med. Res. Opin. 26:
1715-1731.
2. Niethard, F. U., Gold, M. S., Solomon, G. S., Liu, J. M.,
Unkauf, M., Albrecht, H. H. and Elkik, F. 2005. Efficacy
of topical diclofenac diethylamine gel in osteoarthritis of
the knee. J. Rheumatol. 32: 2384-2392.
3. Kulkarni, R. V.,Wagh, Y. J.,Setty, C. M. and Sa, B. 2011.
Development and characterization of sodium alginatehydroxypropyl methylcellulose-polyester multilayered
hydrogel membranes for drug delivery through skin.
Polymer-Plastics Technol. Engin. 50: 490-497.
4. Minghetti, P., Cilurzo, F., Casiraghi, A., Montanari, L.
and Fini, A. 2007. Ex vivo study of transdermal permeation of four diclofenac salts from different vehicles. J.
Pharm. Sci. 96: 814-823.
5. Arora, P. and Mukherjee, B. 2002. Design, development,
physicochemical, and In vitro and In vivo evaluation of
transdermal patches containing diclofenac diethylammonium salt. J. Pharm. Sci. 91: 2089-2096.
6. Meidan, V. M. and Michniak, B. B. 2004. Emerging technologies in transdermal therapeutics. Am. J. Ther. 11:
312-316.
7. Djordjevic, L., Primorac, M., Stupar, M. and Krajisnik,
D. 2004. Characterization of caprylocaproyl macrogolglycerides based microemulsion drug delivery vehicles for
an amphiphilic drug. Int. J. Pharm. 271: 9-11.
8. Kweon, J. H., Chi, S. C. and Park, E. S. 2004. Transdermal delivery of diclofenac using microemulsions.
Arch. Pharm. Res. 27: 351-356.
9. Vucini-Milankovi, N., Savi, S., Vuleta, G. and
Vucini, S. 2007. The physicochemical characterization
and In vitro/In vivo evaluation of natural surfactantsbased emulsions as vehicles for diclofenac diethylamine.
Drug Dev. Ind. Pharm. 33: 221-234.
10. Manosroi, A., Jantrawut, P. and Manosroi, J. 2008. Antiinflammatory activity of gel containing novel elastic
niosomes entrapped with diclofenac diethylammonium.
Int. J. Pharm. 360: 156-163.
11. Khalil, E., Najjar, S. and Sallam, A. 2000. Aqueous

solubility of diclofenac diethylamine in the presence

of pharmaceutical additives: a comparative study with
diclofenac sodium. Drug Dev. Ind. Pharm. 26: 375-381.
12. Baboota, S., Shakeel, F. and Kohli, K. 2006. Formulation
and evaluation of once-a-day transdermal gels of diclofenac diethylamine. Methods Find. Exp. Clin. Pharmacol.
28: 109-114.
13. Zhao, L., Li, Y., Fang, L., Ren, C., Xu, Y. and He, Z.
2009. Effect of O-acylmenthol and salt formation on
the skin permeation of diclofenac acid. Drug Dev. Ind.
Pharm. 35: 814-826.
14. Chaudhary, H., Kohli, K., Amin, S., Rathee, P. and
Kumar, V. 2011. Optimization and formulation design
of gels of Diclofenac and Curcumin for transdermal drug
delivery by Box-Behnken statistical design. J. Pharm.
Sci. 100: 580-593.
15. Yariv, D., Efrat, R., Libster, D., Aserin, A. and Garti,
N. 2010. In vitro permeation of diclofenac salts from
lyotropic liquid crystalline systems. Colloids and Surf B:
Biointerfaces. 78: 185-192.
16. Rubio, L., Alonso, C., Rodriguez, G., Barbosa-Barros, L.,
Cordech, L., De la Maza, A., Parra, J. L. and Lpez, O.
2010. Bicellar systems for In vitro percutaneous absorption of diclofenac. Int. J. Pharm. 386: 108-113.
17. Pathan, I. B. and Setty, C. M. 2009. Chemical Penetration Enhancers for Transdermal Drug Delivery Systems.
Trop. J. Pharm. Res. 8: 173-179.
18. Gupta, G. D. and Gound, R. S. 1999. Release rate of
nimesulide from different gellants. Indian J. Pharm. Sci.
61: 229-230.
19. Meidan, V. M. and Pritchard, D. 2010. A two-layer diffusive model for describing the variability of transdermal
drug permeation. Eur. J. Pharm. Biopharm. 74: 513-517.
20. Shah, S. N. H., Mahboob Rabbani, Muhammad
Fakhruddin Amir. 2005. In vitro study of percutaneous
absorption of diclofenac in the presence of sodium lauryl
sulphate through rabbit skin. J. Res. Sci. 16: 45-50.
21. Ng, S. F., Rouse, J. J., Sanderson, F. D., Meidan, V. and
Eccleston, G. M. 2010. Validation of a static Franz diffusion cell system for In vitro permeation studies. AAPS
PharmSci. Tech. 11: 1432-1441.
22. Adeyemi, O. O., Okpo, S. O. and Ogunti, O. O. 2002.
Analgesic and anti-inflammatory effects of the aqueous
extract of leaves of Persea americana Mill (Lauraceae).
Fitoterapia. 73: 375-380.
23. Bamgbose, S. O. and Noamesi, B. K. 1981. Studies
on cryptolepine II: Inhibition of carrageenan induced
oedema by cryptolepine. Planta Med. 41: 392-396.
24. Sewell, R. D. and Spencer, P. S. 1976. Antinociceptive
activity of narcotic agonist and partial agonist analgesics
and other agents in the tail-immersion test in mice and
rats. Neuropharmacology 15: 683-688.
25. Rice, D. P. and Ketterer, D. J. 1977. Restrainer and cell
for dermal dosing of small laboratory animals. Lab.
Anim. Sci. 27: 72-75.
26. Ngo, M. A. and Maibach, H. I. 2010. Dermatotoxicology:
Historical perspective and advances. Toxicol. Appl.

470
Journal of Food and Drug Analysis, Vol. 20, No. 2, 2012

Pharmacol. 243: 225-238.

27. European Medicines Agency. ICH Topic E 6 (R1) Guideline for Good Clinical Practice. Step 5. Note for guidance
on good clinical practice (CPMP/ICH/135/95). Accessed
on January 2 (2009). www.ema.euro-pa.eu/pdfs/human/
ich/013595en.pdf.
28. World Medical Association Declaration of Helsinki.
Ethical principles of medical research involving human
subjects. Accessed on September 3 (2008). http://www.
wma.net/en/30publications/10policies /b3/index.html.

29. El Maghraby, G. M. M., Williams, A. C. and Barry, B. W.

2001. Skin delivery of 5-fluorouracil from ultradeformable and standard liposomes in-vitro. J. Pharm. Pharmacol. 53:1069-1077.
30. Boelsma, E., Hendriks, H. F. J. and Roza, L. 2001. Nutritional skin care: health effects of micronutrients and fatty
acids. Am. J. Clin. Nutr. 73: 853-864.
31. Rowat, A. C., Kitson, N. and Thewalt, J. L. 2006. Interactions of oleic acid and model stratum corneum membranes
as seen by 2H NMR. Int. J. Pharm. 307: 225-331.