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International Journal of Pharmaceutics 453 (2013) 569578

Contents lists available at SciVerse ScienceDirect

InternationalJournal ofPharmaceutics

journal homepage: www.elsevier .com/ locate / i jpharm

Pharmaceutical nanotechnology

Microemulsion and poloxamer microemulsion-based gel forsustained transdermal delivery ofdiclofenac epolamine using in-skindrug depot: In vitro/in vivo evaluation

Shahinaze A. Fouad a, Emad B. Basalious b,, Mohamed A. El-Nabarawib, Saadia A. Tayel b

a Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy,AhramCanadian University, Cairo,Egyptb Department of Pharmaceutics and Industrial Pharmacy,Faculty of Pharmacy, Cairo University, Kasr El-aini Street, Cairo 11562,Egypt

a r t i c l e i n f o

Article history:

Received 17 April 2013

Accepted 1 June 2013

Available online 18 June 2013

Keywords:

Diclofenac epolamine

In-skin depot

Sustained transdermal delivery

D-optimal design

Microemulsion

a b s t r a c t

Microemulsion (ME) and poloxamer microemulsion-based gel (PMBG) were developed and optimized

to enhance transport ofdiclofenac epolamine (DE) into the skin forming in-skin drug depot for sustained

transdermal delivery ofdrug. D-optimal mixture experimental design was applied to optimize ME that

contains maximum amount ofoil, minimum globule size and optimum drug solubility. Three formulation

variables; the oil phase X1 (Capryol), Smix X2 (a mixture ofLabrasol

/Transcutol, 1:2 w/w) and water

X3 were included in the design. The systems were assessed for drug solubility, globule size and light

absorbance. Following optimization, the values of formulation components (X1 , X2, an d X3) were 30%,

50% and 20%, respectively. The optimized ME and PMBG were assessed for pH, drug content, skin irritation,

stability studies and ex vivo transport in rat skin. Contrary to PMBG and Flector gel, the optimized ME

showed the highest cumulative amount of DE permeated after 8 h and the in vivo anti-inflammatory

efficacy in rat paw edema was sustained to 12 h after removal ofME applied to the skin confirming the

formation of in-skin drug depot. Our results proposed that topical ME formulation, containing higher

fraction ofoil solubilized drug, could be promising for sustained transdermal delivery ofdrug.

2013 Elsevier B.V. All rights reserved.

1. Introduction

Musculoskeletal pain is a common problem often treated

with topical NSAIDs. Topical NSAIDs have a reduced risk of

upper GI complications such as gastric and peptic ulcers, dys-

pepsia as well as a lack of drugdrug interactions (McCarberg

and Argoff, 2010). Diclofenac epolamine is a NSAID, known as

diclofenac-N-(2-hydroxyethyl)-pyrrolidine) (DHEP) (Conte et al.,

2002). The diclofenac molecule, in its acidic form, is hydropho-

bic with very low solubility in water. The epolamine salt of

diclofenac has greater solubility in water and non-polar solvents

(n-octanol) than other diclofenac salts. High concentrations of

aqueous diclofenac epolamine solutions exhibit surfactant behav-ior(McCarberg and Argoff, 2010). The solubility and the surfactant

properties of diclofenac epolamine enhance its membrane perme-

ability (OConnor and Corrigan, 2001).

DE is currently available as topical geland patch marketedunder

the brand name of Flector. Flector patch (10 cm14cm) con-

tains an adhesive material containing 1.3% DE which is applied to a

Corresponding author. Tel.: +20 1200010002.

E-mail addresses:[email protected],

[email protected] (E.B. Basalious).

non-woven polyester felt backing and covered with a polypropyl-

ene film release liner under patch (Petersen and Rovati, 2009). The

use of external drug reservoir (topical patch) is the common tech-

nique used to sustain the transdermal delivery of water soluble

drugs. The major disadvantages of transdermal patches are their

sophisticated method of manufacture and the possibility that a

local irritation will develop at the site of application. Erythema and

itching can be caused by the drug and the adhesive in the patch

formulation. Topical NSAID gels or creams are applied up to four

timesdaily.Moreover, patchesand gelsare inconvenientto patients

regardingdiscrepancy withcleaning and washing of skin. The com-

bination of all advantages of gels (simple method of manufacture

andease of application by patients) with that of patches (sustaineddelivery) is the goal of this study. Our hypothesis was to increase

the penetration of DE through epithelial tissue for loading of the

drug into the skin forming in-skin drug depot where skin acts as

in situ skin patch.

Effective penetration of drugs through the stratum corneum is

a major challenge in transdermal drug delivery. The presence of

lipid of the stratum corneum represents a lipophilic barrier that

restricts the permeation of molecules. Several approaches have

been proposed to increase skin permeation. Microemulsions (MEs)

which are clear, thermodynamically stable mixtures of oil, water

andsurfactant, have been shownto beable to deliver drugsthrough

0378-5173/$ seefrontmatter 2013 Elsevier B.V. All rights reserved.

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570 S.A. Fouad et al./ International Journal of Pharmaceutics 453 (2013) 569578

the skin better than conventional systems such as gel, creams and

ointment (Kreilgaard, 2002). ME and ME based gels were pre-

pared in an attempt to increase the transdermal drug delivery of

both hydrophilic and lipophilicdrugs (Barot et al., 2012; Kreilgaard

et al., 2000; Trotta et al., 1997). Moreover, NSAIDs are one of

the most important drug classes that have been formulated as

microemulsion-based hydrogels for both topical and transdermal

use such as ibuprofen (Chen et al., 2006), ketoprofen (Rhee et al.,

2001) and diclofenac (Kweon et al., 2004).

MEs and ME based gels were found to have favorable solvent

properties due to the potential incorporation of large fraction of

lipophilic and/or hydrophilic phases (Malcolmson and Lawrence,

1993; Malcolmson et al., 1998). Only the dissolved fraction of a

drug in a vehicle can enter the skin. The small globule size of MEs

makes them a suitable vehicle to penetrateepithelial tissueand use

skin as a depot forsustained drug delivery (Yuan andAcosta,2009).

Development of a pharmaceutical formulation consumes a lot

of time and is considered as a complex process. Thus, D-optimal

mixture design is applied to develop pharmaceutical formulation

because it was demonstrated to be an efficient method for opti-

mization of the formulation and to understand the relationship

between independent variables and dependent variables in a for-

mulation (Basalious et al., 2010; Gao et al., 2004).

Literature lacks any data about the use of ME for loading of druginto skin to form in-skin depot for sustained transdermal delivery

ofDE (awater soluble drug).Thus,the aim of this study was the for-

mulation and optimization of ME and PMBG. D-optimal design was

applied to optimize formulation that contains a maximum amount

of lipid, small globule size (

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S.A. Fouad et al./ International Journal of Pharmaceutics 453 (2013) 569578 571

Table 1

The formulations of mixture design and their characterization results.

Formulation A: Capryol B: Smix C: Water Solubility (mg/ml) Globule size (nm) Absorbance

1 20 70 10 292.27 62.93 0.075

2 10 70 20 268.61 49.15 0.023

3 30 50 20 374.70 77.2 0.045

4 30 50 20 277.04 28.19 0.01

5 25 60 15 188.29 48.9 0.032

6 15 65 20 248.00 37.55 0.012

7 10 60 30 304.44 54.8 0.0348 30 60 10 262.29 50.4 0.036

9 10 80 10 171.19 151.6 0.089

10 30 60 10 260.42 60.5 0.027

11 20 50 30 358.31 43.45 0.059

12 22.5 55 22.5 273.77 54.6 0.022

13 15 55 30 480.09 51.6 0.023

14 10 60 30 359.09 59.6 0.024

15 20 50 30 370.02 54.68 0.025

16 10 80 10 248.71 140.6 0.091

of Smix, intermediate drug solubility (Y1) and the globule size (Y2)

less than 100 nm.

2.5. Preparation of MEs

Fromthe pseudo-ternaryphase diagrams,Smix ratio withmaxi-

mum ME regionwas selected. Different proportions of oiland Smix

were mixed based on the ratios presented in Table 1. The mixture

of oil and Smix was mixed using vortex (VSM-3 model, PRO Scien-

tific Inc., Oxford, England) at ambient temperature. The measured

amount of distilled water was added drop wise to the oily mixture

until clear and transparent liquid was obtained. All MEs were then

stored at ambient temperature.

2.6. Evaluation of the prepared MEs

2.6.1. Determination of drug solubility in the prepared MEs

The solubility of DE in MEs (in mg/ml: Y1) was determined.

Excess amount of DE was added in 5g of each of the previouslyprepared ME in 10-ml-capacity stoppered vials. The resultant mix-

ture was mixed initially by vortex mixer then, all the vials were

shaken in the shaker for 24h at 25C.

Afterwards, centrifugationwas done at 4000 rpmfor 10min and

the concentration of DE in the supernatant was determined by UV

spectrophotometer after appropriate dilution with methanol at its

respective max. The plain ME without drug with the same compo-

sitionwas taken as blank after appropriate dilution with methanol.

2.6.2. Determination of globule size by photon correlation

spectroscopy

The globule size (in nm: Y2), was determined using photon

correlation spectroscopy that analyzes the fluctuations in light

scattering due to the Brownian motion of particles using MalvernZetasizer Nano-ZS (Ver.6.20, Malvern Instruments Ltd., Worcester-

shire, England). All measurements were done at room temperature

(25 C)and at90 C to the incident beam.

2.6.3. Measurement of spectroscopic absorbance at 400nm

The optical clarity of aqueous dispersions of SNEDD formu-

lations was measured spectroscopically. The absorbance of each

formulation was measured at 400 nm, using distilled water as a

blank.

2.6.4. Transmission electron microscopy (TEM) of the optimized

DE loadedMEs

The morphology of the optimized ME systems was observed

using transmission electron microscopy. A drop of each ME was

placed on a copper grid and the excess was removed with a fil-

ter paper. One drop of 2% aqueous solution of phosphotungistic

acid (PTA) was added onto the grid and left for 3060s to allow

staining. The excess was removed with a filter paper. The grid was

finally examinedunder the transmission electron microscope (JEOL

(JEM-1400), Tokyo, Japan).

2.7. Formulation of DE-loaded ME and PMBG

As MEs have low viscosity, their retention at the affected parts

is quiet less. Therefore, their viscosity was required to be increased

by the addition of a suitable gelling agent. Poloxamer was used

as a gelling agent for the optimized ME formulation to formulate

thermosensitive microemulsion-based gel of DE.

Plain poloxamer gel (25%) was firstly prepared according to the

cold technique (Chang et al., 2002; Shin et al., 1999). ME contain-

ing the drug was added portion-wise onto the plain gel in a ratio

of gel:ME (2:1) with continuous stirring. The final microemulsion-

based gel formulation contained 1.3% w/w DE. DE was dissolved

directly in the optimizedME to prepare drug loaded ME containing

1.3% w/w DE.

2.8. Evaluation of DE microemulsion and PMBG

2.8.1. pH measurements and drug content

The apparent pH of the formulations was measured by a pH

meter in triplicate at 25C. For determination of drug content, one

gram of ME formulations was diluted with appropriate amount of

methanol. The concentration of DE was determined by UV spec-

trophotometer at its respective max. The plain ME formulations

without drug with the same composition was taken as blank after

appropriate dilution with methanol.

2.8.2. Stability study

The optimized DE loaded ME and PMBGl were stored at

40 C/75% RH for three months. Optical clarity and drug content

were performedfor thestored drug loadedME and microemulsion-

based gelusingthe same procedures adopted forthe fresh samples.

Morphology of the stored drug-loaded ME was determined using

transmission electron microscopy.

2.8.3. Skin irritation test

Three male albino Wistar rats (130150g) were kept under

standardlaboratoryconditionsand housed in cages withfree access

to a standard laboratory diet and water ad libitum. A single dose of

100L of the optimized drug-loaded ME, optimized drug-loaded

PMBG and the market formulation (Flector

gel) was applied to the

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left ear of the rat, with the right ear as a control. The development

of erythema was monitored for 24h then the gel was removed,

and the application sites were graded according to a visual scoring

scale from no erythma, mild, moderate, high and severe erythema

(Azeem et al., 2009; Shakeel et al., 2007).

2.8.4. Study of ex vivo transport of DE from optimized

formulations into rat skin and ability to form in situ drug depot

Ex vivo skin transportstudies were performedusingnewly bornrat skin (Azeem et al., 2009; Sarigullu Ozguney et al., 2006). Newly

born albino Wistar rats were sacrificed and skin samples obtained

was inspected for the presence of any holes or irregularities. Fresh

skin used in the study was preserved in 10% glycerin solution

at 20 C. The study performed in this section was approved by

Research Ethics Committee, Faculty of Pharmacy, Cairo University.

Skin was slowly thawed and was cut into small circular pieces. The

lower surface of the skin was allowed to hydrate for 1h at 37C

prior to experimentation.

The Ex vivo skin transport studies of DE from the optimized ME,

PMBG and the market product (Flector gel) were performed in a

USP dissolution apparatus tester (USP apparatus II) at 370.1 C.

One gram of drug loadedME, PMBGand the market formulation,

all containing 1.3% drug w/w were placed in double open-sidedglass cylindrical tubes (2.5 cm in diameter and 5 cm in length, with

area=4.9cm2) tightly covered from one side with rat skin. The

loaded tubes were attached from the second side to the shafts of

the USP dissolution tester apparatus. This assembly represents the

donor compartment. The shafts rotated at a speed of 50rpm in

phosphate buffer pH 7.4. The dissolution vessels (receptor com-

partment) were filled with 300 ml of phosphate buffer pH 7.4.

Four milliliter samples were withdrawn periodically at pre-

determined time intervals of 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7

and 8 h and replaced instantly by equal amount of fresh phosphate

buffer pH 7.4 in order to maintain the same volume.

The drug concentration was determined by UV spectropho-

tometer at 276n m. The skin transport studies were done in

duplicates and the average percentage drugpermeated was plottedversus time. Cumulative amount of drug in receptor chamber for

the three formulations was plotted as a function of time.

To study the ability of the three formulations to form in situ

depot in the skin, transport of DE in the skin was observed after

removal of the formulationsfrom thedonorcompartment. Ratskin

was removed after 3 h. The formulations were wiped off with wet-

ted cotton pieces then the rat skin was mounted again on glass

cylinder to continue the skin transport study.

2.8.5. In vivo study of the anti-inflammatory efficacy and

sustained delivery of in-skin depot of DE

The sustained anti-inflammatory efficacy and the ability of the

optimized ME and PMBG to form in-skin depot were compared

in vivo using carrageenan induced rat pawedema test. Flector gelis the marketformulationand it is used as a reference product. Also,

plain PMBG is prepared to be used as a control. Each formulation

except the prepared plain gel contains 1.3% w/w DE.

Thirty two adult male albino Wistar rats, weighing 130150 g

were used in this study. They were purchased from Helwans Farm

of experimental animals (Cairo, Egypt). The animals were accli-

matized to environment for one week, they were housed under

controlled environment at 251 C with a 12hur light/dark cycle.

All animals had free access to standard rodent pellet food consist-

ing of vitamin mixture (1%), mineral mixture (4%), corn oil (10%),

sucrose (20%), cellulose (0.2%), casein 95% (10.5%), starch (54.3%)

and water.

Animals were divided into four groups of eight rats each. The

plain PMBG was assigned to the first group, the optimized ME was

assignedto thesecond group, PMBGwas assignedtothethirdgroup

and the Flector gel was assigned to the fourth group.

In order to induce inflammation,animalswere firstinjected with

0.1ml of 1% carrageenan solution in saline in the plantar region of

the right hind paw. The initial paw thickness (Ti) was measured

using a Micrometer Caliper, one hour after carrageenan injection.

Then, 1g of each formulation was applied to the right hind paw

of the rats. After 3 h of formulation application (sufficient time for

skin loading and formation of in-skin drug depot), formulations

remaining on the surface of the paw were wiped off with cotton

then, the paw thickness (Tf) was measured again using a Microm-

eter Caliper at different time intervals (3, 4, 5, 6, 7, 8 and 12h). The

edema % was calculated from the mean effect in treated animals

according to the following equation:

% edema =Tf Ti

Ti 100

where Tf is the thickness measured following administration of the

formulae at different time intervals. Ti is the thickness measured

1 h after carrageenan sodium injection. Data were analyzed statis-

tically by Students t-test at 5% significance level using GraphPad

Prism 5 program (GraphPad Inc., USA).

3. Results and discussion

3.1. Screening of components for ME

Thesaturated solubility of DE in various oils, surfactants andco-

solvents was estimatedas shown in Fig.1. Amongst the various oily

phases that were screened, Capryol 90 provided the highest solu-

bilityof DEso was chosen for further investigations. Solubilityof DE

in Labrasol was the highest among the surfactants. Labrasol was

selected for further studies due to its solubility profile and its low

toxicity levelas a non-ionicsurfactant (Shafiq-un-Nabi et al., 2007).

Transcutol HP, which is a solubilizer and absorption enhancer

(Basalious et al., 2010), was found to be a very efficient solubilizer

for DE, and so was chosen as a co-solvent in the development of

DE loaded ME formulations aiming to improve the drug loadingcapabilities.

3.2. Construction of pseudo-ternary phase diagrams

To obtain the appropriate components and their concentration

ranges for MEs, pseudo-ternary phase diagrams were constructed

for different Smix ratios 1:1, 1:2 and 2:1, so that o/w ME regions

could be identified and ME formulations could be optimized.

The three ratios gave stable and clear MEs but the ratio which

gave the largest ME region was found to be 1:2 and therefore it

was selected for further studies. This is clearly shown in Fig. 2. The

phase study clearly reveals that with a decrease in the weight ratio

of Labrasol from 1 to 0.5, the ME region is expanded. This obser-

vation conforms to the results obtained from the study ofBarotet al. (2012). It is obvious also thatan increase ofthe weightratio of

Labrasol from 1 to 2 resulted also in expansion of the ME region.

This observation is in agreement with Shakeel et al. stating that

as the surfactant concentration was increased in the Smix ratio, a

higher ME region was observed, perhaps because of further reduc-

tionof theinterfacial tension,increasingthe fluidity of theinterface,

thereby increasing the entropy of the system (Shakeel et al., 2007).

Thus, the effect of Labrasol on ME area depends the other com-

ponents of ME especially co-solvent. This is because the reduction

of o/w interface is not achieved by single-chain surfactants alone.

The combination of short to medium chainlength alcohols (such as

Transcutol HP) withsingle chain surfactants couldresult in lower-

ing the interfacial tension due to increased fluidity at the interface

(Binks et al., 1989). Miscibility of aqueous and oily phases could

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Fig. 1. Solubility (mg/ml) of diclofenac epolamine in various microemulsion components.

Fig. 2. Pseudo-ternary phasediagrams of microemulsionscomposedof oil (Capryol

90), Smix(surfactant: Labrasol

, co-solvent: Transcutol

) andwaterat various oil/Smixratios 1:2 (a),1:1 (b) and 2:1 (c).

also be increased by medium chain length alcohols due to their

partitioning behavior between the two phases (Lawrence and Rees,

2000; Shafiq-un-Nabi et al., 2007).

3.3. Formulation optimization of MEusing D-optimal design

In order to rapidly obtain the optimal ME, D-optimal mixture

experimental design was applied in this study. The oil phase X1(Capryol 90), Smix X2 (a mixture of Labrasol

/Transcutol, 1:2

w/w) and aqueous phase X3 (water) were chosen as formulation

variables and the solubility of drug in ME, mg/ml (Y1), mean glob-

ule size(Y2) and absorbance of ME (Y3) were used as the responses

(dependent variables). The responses of these formulations are

summarized in Table 1. The independent and response variables

were related using polynomial equation with statistical analysis

through Design-Expert software. As shownin Table 2, the approx-

imation of response values ofY1 based on linear model was the

most suitable because its PRESS was smallest. The values of the

coefficients X1, X2 and X3 are related to the effectof these variables

on the response. A positive signof coefficient indicates a synergistic

effect while a negative term indicates an antagonistic effect upon

the response (Huang et al., 2005). The larger coefficient means the

independent variable has more potent influence on the response.

As shown in Table 1, solubility of DE in the different ME formu-

lation varied between 171.1 and 480mg/ml. It can be inferred that

the three independent factors have a profound effect on drug sol-

ubility. As illustrated in Table 3, a p-value of0.05 for any factor

in analysis of variance (ANOVA) indicates a significant effect of the

corresponding factors on the solubility of drug in ME (Y1). It can

be inferred that the terms X1, X2, and X3 have a significant effecton the drug solubility (p< 0.05). This result could be confirmed by

the positive value of these coefficients (Table 2). Fig. 3 shows the

contour diagrams illustrating the effect of varying ratios of (X1),

(X2) and(X3) on the solubility of drug in ME (Y1). It is obvious that

the water content in ME formulation has the highest positive effect

on the solubility of DE in ME. This means that increasing the water

Table 2

Reduced Regression results of the measured responses.

Response Model R2 Adjusted R2 Predicted R2 PRESS Regression e quation f or t he r esponses

Y1 Linear 0.547 0.4774 0.342 58,715.09 Y1 = +307.68X1 + 117.74X2 + 832.50X3Y2 Quadratic 0.8193 0.7537 0.5469 7792.269 Y2 = +526.53X1 + 384.48X2 + 830.97X3 1631.31X1X2 2172.43X2X3Y3 Quadratic 0.7951 0.7439 0.6327 0.0036 Y3 =0.29X1 +0.26X2 +1.25X3 2.70X2X3

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Table 3

ANOVA of thesolubility of drug in microemulsion formulations(Y1).

Source Sum of squares dF Mean square F p-Value

Model 48,819.5 2 24,409.7 7.85143 0.0058

Linear Mixture 48,819.5 2 24,409.7 7.85143 0.0058

Residual 40,416.4 13 3108.95

Lack of Fit 31,080 8 3885 2.08057 0.2179

Pure Error 9336.38 5 1867.28

Cor Total 89,235.8 15

Fig. 3. Contour plot ofthe effectof variableson thesolubilityof drug in microemul-

sion formulations (Y1).

content in ME formulation increases the fraction of DE that is sol-uble in the aqueous phase of ME.

As shown in Table 1, the globule size of the different ME for-

mulation varied between 28.19 and151.6nm. As shown in Table 2,

the approximation of response values ofY2 and Y3 based on the

quadratic model was the most suitable.

ANOVA of the effect of variables on globule size (nm) of ME (Y2)

and spectroscopic absorbance of ME (Y3) shows that the terms X1,

X2, and X3 have a significant effect on both responses (p< 0.05).

Thecoefficient of X2X3 for both responses was largest, showing the

negative effect of combination of Smix and water content on the

globule size of the MEs and thus, their absorbance. Fig. 4a and b

shows the contour diagrams illustrating the effect of varying ratios

of(X1),(X2) and (X3) on the globule size (nm)of ME (Y2) and spec-

troscopic absorbance of ME (Y3), respectively. It is obvious that

there is an optimum ratio of all the mixture components for ME

formulation having small globule size and absorbance. Sufficient

concentration of water is needed for maximal effect of Smix on

emulsification of Capryol.

The aim of the optimization of pharmaceutical formulations

is generally to determine the levels of the variable from which a

robust product with high quality characteristics may be produced

(Basalious et al., 2010). Some of the measured responses have to

be minimized. In this case, these responses comprise the globule

size (

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Fig. 5. Overlay plot for the effect of different variables on the three responses.

The solubility of drug in microemulsion, mg/ml (Y1), mean globule size (Y2) and

absorbance of microemulsion (Y3

).

3.4. Stability studies of the optimized formulations

The optimized formulations were stable when stored at

40 C/75% RH for three months where there was no obvious change

in visual appearance. The drug content of the fresh and stored for-

mulations of the optimized drug-loaded ME were 100.62%2.12

and 98.54%2.82, respectively. The drug content of the fresh and

stored formulations of the optimized PMBG were 104.89%3.22

and 101.98%3.6, respectively. The pH values of the fresh and

stored optimized formulations ranged from 6.5 to 7. The morphol-

ogy of the optimized drug-loaded ME examined via TEM was not

changed before and after storage. Transmission electron micro-graphs of the optimized formulation, Fig. 6 revealed that the

globules of the developed MEs are spherical, discrete and have

uniform droplet size distribution. Globules appear to have compa-

rable size to the calculated values obtained by photon correlation

spectroscopy (Table 1).

3.5. Skin irritation test

Various formulations, when applied topically, might cause skin

irritation. Thus, rat skin irritation experiments were conducted in

order to assess the potential irritant effects of the optimized drug-

loaded ME, PMBGand the market formulation (Flector gel).All the

ME formulations and the market product show no erythema. Thus,

the optimized ME formulations were safe to be used for transder-

mal drug delivery.

3.6. Ex vivo transport of DE from optimized formulations into rat

skin and ability to form in situ drug depot

Ex vivo skin transport from the optimized drug-loaded ME,

PMBG and the market formulation (Flector gel) through newly

born rat skin are illustrated in Fig. 7.

One gram of each formulation was placed on skin of newly

born rat attached to a cylindrical tube having surface area 4.9 cm2.

Amongst the formulations tested, the optimized drug-loaded ME

showed the highest cumulative amount of DE permeated after

8 h (345.45g/cm2 29.8) followed by PMBG (57.45g/cm2 9.8)

and finally the market formulation (9.45g/cm2 2.9). The con-

tent of the surfactants mixture in MEs significantly enhanced the

transport of drug through skin. Moreover, the small globule size

of the ME droplets also affects the percutaneous absorption of the

drug. When the droplet size is very small, there is a chance that the

number of vesicles that can interact with a fixed area of stratum

corneum to increase, thereby increasing the efficiency in percuta-

neous uptake(Shah et al., 2010). Thus, thehigh skin transportof DE

from ME is mainlydue to the amount of drug solubilized in small oil

globules thateasily transport through the lipidof stratum corneum

of the skin. Although containing the same surfactant mixture and

globule size as ME, PMBG showed significant reduction in drug

transport in skin. An explanation for this observation may be due

to the high water content of PMBG (about 75%). The major amount

of DE is located in the aqueous phase interacting with Poloxamer

micelles and consequently lower transport rate through the skinwas observed (Xuan, 2011). The explanation is useful also for the

poor skin transport of DE from Flector gel compared with ME for-

mulations especially when we know that composition of Flector

gel contains about 75% water with nonionic surfactant such as PEG

400 monostearate. The lack of oil globules in the market product

Fig. 6. Transmission electron micrographs of the fresh (a)and stored(b) optimized drug-loaded microemulsion taken at 30,000 magnification.

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Fig. 7. Permeation profiles of DE from ME, PMBG and Flector gel through rat skin.

explains the significant lowering of DE skin transport compared to

PMBG.

To confirm that DE containing oil globules transport through

stratum corneum and act as in-skin drug depot for sustained deliv-

ery of drug, release of DE from the skin was observed after removal

of formulations after 3 h of permeation from the donor compart-

ment. As shown in Fig. 7, the cumulative amount of DE permeated

from ME (removed 3 h after permeation from donor compartment)

was continuously increased with lower rate compared with that

of normal ME. This observation confirms that skin acts as drug

reservoir (in-skin depot) after removal of ME. In case of PMBG

and Flector gel, there was no remarkable increase of the cumula-

tive amount permeated of DE after removal of these formulations

from donor compartment. Thus, the gel matrix rather than skin

acts as drug reservoir for these formulations. Contrary to ME, the

availability of the gel on skin surface is very important to main-

tain anti-inflammatory efficacy. To confirm the previous results,

the three formulations were subjected to in vivo study for treating

inflamed rat skin.

3.7. In vivo study of the anti-inflammatory efficacy and sustained

delivery of in-skin depot of DE

The anti-inflammatory efficacy of DE was taken as a measure

of in-skin depot formation and the extent of transport of drug

through the skin from the medicated formulations (ME, PMBG and

Flector gel). After 3 h of formulation application (sufficient time

for skin loading and formation of in-skin drug depot), formulationsremaining on the surface of the paw were wiped off with cotton.

Rat hind paw edema was used as a model for inflammation

in this study (Winter et al., 1963). Results revealed that injection

of carrageenan (selected as inflammagen) produced a pronounced

edema. Formulations applied to inflamed area were removed after

3h of application. Thus, the higher the amount of DE loaded into

skin (in-skin drug depot), the extended transdermal drug delivery

and the higher is the anti-inflammatory efficacy as the skin itself

acts as drug reservoir.

The anti-inflammatory efficacy of single dose application of

DE-loaded ME and PMBG was testedcompared to Flector gel con-

tainingthe sameconcentration on thecarrageenaninducedrat hind

paw edema at different time intervals up to 12h using plain base

as a control. As shown in Fig. 8, the inhibition of edema started 5 h

Fig. 8. Anti-inflammatory efficacy of drug-loaded ME and PMBG compared to the

marketproduct in rat paw edema.

after formulation application (2h after formulation removal). The

highest inhibition of edemawas observedin caseof drug-loaded ME

where the effect was sustained to 12h and was significantly differ-

entfromthatofplainbase 6 h (p< 0.05). Fig.9 shows photoimagesof right hind rat paw showing edema before and six hours after

application of drug-loaded microemulsion. These results correlate

well with results previously obtained by ex vivo skin transport

study confirming that oil globules of ME containing solubilized DE

penetrate through stratum corneum and act as in-skin drug depot

for the sustained delivery of drug in the skin. This is highly useful

in dermal andtransdermal deliver of drugs where the delivery sys-

tem is applied onto skin for few hours (at night before sleeping)

to load skin with drug then the in-skin depot continue the trans-

dermal delivery of the drug. This is advantageous in case of skin

where topical application of conventional systems for treatment

of soft tissue injuries is highly frequent reaching up to four times

daily. The inhibitory effect of PMBG and Flector gel was remark-

ably lower than that of ME confirming that these gels act as the

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Fig. 9. Photoimages of right hind rat paw showing edema beforeand 6 h after application of drug-loaded microemulsion.

drug reservoir and its availability on the skin surface is a must to

sustain the delivery of the drug into the skin.

4. Conclusion

In this study, ME and PMBG of DE were prepared and in vitro

evaluated. D-optimal mixture experimental design was applied in

order to rapidly obtain the optimal DE-loaded ME formulations

containing maximum amount of oil having minimum globule size

which allow transport of drug into skin forming in-skin depot

for sustained transdermal delivery of the drug. The optimized ME

formulation composed of 30% Capryol, 50% Smix (a mixture of

Labrasol/Transcutol, 1:2w/w)and 20% water. Thestabilityof the

optimized formulation was retained after storage at 40 C/75% RH

for three months. The ME formulations showed no skin irritation

and are safe to be used for transdermal drug delivery. Contrary

to PMBG and Flector gel, the optimized ME showed the highest

cumulative amountof DE permeated after 8 h andthe release of DEfrom the skin was observed even after removal of ME applied to the

skin. The high skin transport of DE from ME is mainly due to the

amount of drug solubilized in small oil globules that easily trans-

port through the lipid of stratum corneum of the skin. The in vivo

anti-inflammatory efficacy in rat paw edema was sustained after

removal of ME applied to the skin confirming the formation of in-

skin drug depot. The significant increase in DE transport through

the skin and the formation of in-skin drug depot by the developed

ME propose that theprepared systemcould be promisingto sustain

the transdermal delivery of DE for treatment of soft tissue injuries.

The extended transdermal delivery of the optimized ME and clini-

cal evaluation on human patients with musculoskeletal pain needs

to be investigated.

Acknowledgements

We are very grateful for Marcyrl for Pharmaceutical Industries

and Gattefosse for providing the required chemicals for research

work. We are also grateful to Dr. Ayman El-Sahar (Department of

Pharmacology and Toxicology, Faculty of Pharmacy, Cairo Univer-

sity), for his kind help in the in vivo study in this paper.

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