developing an efficacious diclofenac diethylamine transdermal formulation.pdf

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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. SIPBS, University of Strathclyde, Glasgow, Scotland, UK

5. 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) concen-tration 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-inflamma-tory drug (NSAID) that is widely used to symptomatically alleviate the pain and swelling associated with conditions such as arthritis, toothache, dysmenorhea and other muscu-loskeletal 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 consider-able 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 diethyl-ammonium salt of this NSAID. In terms of physicochemical

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). Phar-macologically, 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 aggre-gates termed bicelles(16).

Chemical substances temporarily diminishing the barrier of the skin and known as accelerants or sorption

* Author for correspondence. Mobile: 00923142082826; Fax: 0092992383441; Email: [email protected]

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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 acid-turpentine 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 anti-inflammatory, 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 incor-porated 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, Swit-zerland). 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.

II. Preparation of Diclofenac Lotions

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

(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 previ-ously(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/tWhere

S = SpreadabilityM = Weight/Volumes tide to upper slideL = Length of glass slidet = Time taken to separate the slides completely from

each otherEach formulated lotion was evaluated for homoge-

neity 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 month-long 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).

V. HPLC Assay

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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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 propyl-paraben showed a retention time of 4.1 min. Intra- and inter-day 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 coef-ficient (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 excipi-ents 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 stir-ring 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 concentra-tion values being mathematically corrected for the progres-sive dilutions caused by repeated sampling. Sink conditions existed throughout the experiment. Since skin exhibits large sample-to-sample permeability differences(19), each experi-ment 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 carrageenan-induced rat paw edema assay(22). The assay was run on male Wistar rats (150 5 g) purchased from the Institute of Biotech-nology 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 inflamma-tion was quantified by measuring the linear paw circumfer-ence(23). Each test lotion was applied to 3 rabbits.

In the next set of in vivo studies, each analgesic-containing lotion was evaluated for its antinociception effect by running a modified version of the established hot water-tail 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 admin-istration, 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 subse-quent 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 cuta-neous 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 Cauca-sian 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 sensa-tory 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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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 Depart-mental Ethical Committee and were conducted according to the guidelines of Good Clinical Practice(27) and Helsinkis Declaration regarding human use in experiments(28).

IX. Statistics

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. Statis-tical 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 signifi-cant (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 perme-ation 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 sili-cone membrane material undergoing perturbations, such as perhaps adsorption of excipients, upon initial exposure to the

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 stan-dard 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

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Figure 1. Cumulative permeation of DDA across silicone membrane as a function of time. Error bars represent SD values, with n = 5.

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0 500 1000 1500Time (min)

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


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g/cm2/min. In contrast, L1, L2, L3 and L4 exhibited respec-tive 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 inflam-mation. Another noteworthy point is that while L1 was signifi-cantly (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

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 antinoci-ception 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 accept-able 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.

Table 1. In vivo edema reduction induced by each DDA formulation in carrageenan-challenged rabbits (Mean SD, n = 3)

FormulatedLotion

Anti-inflammatory activity

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-sig-nificant changes.

Table 2. In vivo tail flick response times (antinociception) associated with each DDA formulation at 30, 45 and 60 min after lotion applica-tion (Mean SD, n = 3)

FormulatedLotion

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 12.14 2.1 13.69 2.2

LC 1.39 0.76 1.75 0.87 2.19 0.93


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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).

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