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This article presents the development and evaluation of a new topical formulation of diclofenac diethylamine (DDA) as a locally applied analgesic lotion. To this end, the lotion formulations were formulated with equal volume of varying concentrations (1%, 2%, 3%, 4%; v/v) of propylene glycol (PG) and turpentine oil (TO) as permeation enhancers. These lotions were subjected to physical studies (pH, viscosity, spreadability, homogeneity, and accelerated stability), in vitro permeation, in vivo animal studies and sensatory perception testing. In vitro permeation of DDA from lotion formulations was evaluated across rabbit and polydimethylsiloxane membrane using Franz cells. It was found that PG and TO content influenced the permeation of DDA across model membranes with the lotion containing 4% v/v PG and TO content showed maximum permeation enhancement of DDA. In the in vivo animal testing, lotion with 4% v/v enhancer content showed maximum anti-inflammatory and analgesic effect without inducing any irritation. Sensatory perception tests involving healthy volunteers rated the formulations between 3 and 4 (values ranging between -4 to +4, indicating very bad to excellent respectively). It was concluded that the DDA lotion containing 4% v/v PG and TO exhibited the best performance overall and that this specific formulation should be the basis for further clinical investigation.
Keywords: Diclofenac diethylamine, lotion, propylene glycol, percutaneous absorption, turpentine oil, topical.
Diclofenac is an important member of a class of drugs known as nonsteroidal anti-inflammatory drugs (NSAID) which is widely used for the treatment of musculoskeletal disorders, arthritis, toothache, dysmenorrhea and symptomatically relief of pain and inflammation . Diclofenac diehtylamine (DDA) has limited bioavailability (40-60%), a short half-life (2-3 h) and low therapeutic dose requirement (25-50 mg) [1, 2]. The dose-dependent gastrointestinal, cardiovascular and renal unwanted effects following oral delivery of NSAID's has promoted its transdermal delivery  which has several advantages over oral delivery to improve patient compliance . In recent years, extensive research has been carried out to curtail the barrier properties of stratum corneum which compromises the percutaneous absorption using permeation enhancers [5-9]. Research has been carried out to improve percutaneous absorption of DDA involving various techniques including gel [1, 10], microemulsion , liposomes [12, 13], lyotropic liquid crystal  and drug-excipient combination [2, 14] based formulations.
Natural products including essential oils [15-17] are gaining importance as permeation enhancers in transdermal drug delivery owing to their good safety profile . Turpentine oil (TO) has been used as penetration enhancer for a number of hydrophilic and lipophilic drugs [19, 20]. TO contain terpenes which are less toxic and FDA has classified terpenes as 'generally recognized as safe' (GRAS) . Propylene glycol (PG) has been widely used as solvent and permeation enhancer in various transdermal formulations [22, 23].
In the present work we investigated percutaneous delivery of a new topical DDA formulation containing PG and TO as permeation enhancers. These formulations were characterized for its pH, viscosity, spreadability and homogeneity, accelerated stability, in vitro skin permeation across two model membranes, namely polydimethylsiloxane membrane and rabbit skin. In vivo evaluations included animal models and human volunteer's sensatory perception test.
Propylene glycol (Merck, Germany); ethanol (Merck, Germany); sodium acetate (Merck, Germany); isopropyl alcohol (Fluka, Switzerland); carbomer 980 (Fisher, Germany); É£-carrageenan No. 2249 (Fluka Biochemika, Switzerland); Turpentine oil (MS Traders, China); diclofenac diethylamine (Novartis, Pakistan); polydimethylsiloxane membranes with 400 Î¼m-thickness (Samco, USA) silicone grease (Dow Corning, USA); sodium chloride (Merck, Germany).
Preparation of lotion formulations
All the lotion formulations were prepared as described in the Table I.
Table I. DDA containing lotion formulations
Carbomer 980 (mg)
Phosphate buffered saline (mL)
(% v/v of total lotion )
(% v/v of total lotion)
Ethanol (q.s. for 100 mL lotion)
Diclofenac diethylamine quantification
The amount of drug was quantified using a Waters UV/Vis HPLC system installed with a symmetry C18 reverse phase column (5Âµm, 4.6 Ã- 25cm) (Waters, UK) with UV detection set at 276 nm. The samples were injected with a rheodyne injector having a 20 ÂµL loop volume. The elution was carried out at ambient temperature and an isocratic mobile phase composed of methanol and 0.1M sodium acetate (70:30 v/v) with a flow rate of 0.8 mL/min was used for separation. The mobile phase was prepared on daily bases and it was filtered and then degassed prior to use. The method was validated as per ICH guidelines with precision (less than 1% RSD) and % accuracy (% RSD 0.865). The limit of detection was 225.2 ng/mL and the limit of quantitation was 350.7 ng/mL for DDA. DDA solutions of known concentrations were used to obtain a standard calibration curve.
In vitro characterisation of lotion formulation
pH and rheological measurements
Lotion pH was recorder with a digital pH meter (Mettler & Toledo, Giessen, Germany) by inserting probe into the lotion formulation and allowing it to equilibrate for 1 minute. Viscosity measurements were conducted using a Model RVTDV II Brookfield viscometer (Stoughton, MA). A C-50 spindle was employed, with a rotation rate of 220 rpm. The gap value was set to 0.3 mm. Temperature was set at 25Â°C Â± 2 and these experiments were conducted in triplicate to obtain statistically significant data.
Spreadability and homogeneity determination
The spreadability of each lotion was determined by the wooden block and glass slide method previously detailed somewhere else . Essentially, a 5mL volume (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 slides was noted. Spreadability was determined according to the formula:
S = Spreadability expressed in mg.cm.sec-1.
M = Weight/Volumes tide to upper slide (mg)
L = Length of glass slide
t = Time taken to separate the slide completely from each other
Again experiments were repeated three times to obtain a statistically significant data.
Each formulated lotion was evaluated for homogeneity by naked eye examination. This involved a subjective assessment of appearance including the presence of any aggregates.
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 Â± 2 °C, 75% relative humidity. 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).
White New Zealand male rabbits weighing between 3-4 kg were used for the preparation of skin. The skin samples were excised from the abdomen region. Hairs were clipped short and adhering subcutaneous fat was removed carefully from the isolated full thickness skin. Then, the skin was cut into samples that were just larger than the surface area of the Franz diffusion cells. To remove extraneous debris and any leachable enzyme, the dermal side of the skin was kept in contact with a normal saline solution for 1 hour prior to start the diffusion experiments. For the polydimethylsiloxane membrane studies, pieces of a size suitable for mounting in Franz cells were cut out and then soaked overnight in PBS (pH 7.4). This procedure was performed in order to allow the removal of excipients present within the membrane upon purchase .
Permeation experiments were performed using Franz cells manufactured 'in house', exhibiting a diffusional area of 0.85cm2 and a receptor cell volume of 4.5 mL. Subsequently, the test membrane (either rabbit skin or polydimethylsiloxane) was inserted as a barrier between the donor and receiver cells. Silicone grease was applied in order 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 while receptor compartment was filled with PBS maintained at pH 7.4 which is close to the pH of blood . The diffusion cells were placed on a stirring bed (Variomag, US) immersed in a water bath at 37 Â± 5Â°C to maintain a temperature of ~32Â°C at the membrane surface. At scheduled times, a 0.5 mL aliquot of receiver fluid was withdrawn and the receiver phase was replenished with 0.5 mL of fresh pre-thermostated PBS. Withdrawn aliquots were assayed immediately by HPLC for DDA quantification. Sink conditions existed throughout. Since skin exhibits large sample-to-sample permeability differences , therefore, each experiment consisted of 5 replicate runs (n=5).
In vivo characterization
The in vivo research consisted of three separate types of studies. All these were conducted under conditions that had been regulated and approved by the Animals Ethics Committee of Bahauddin Zakariya University (Pakistan).
Each DDA-containing formulation was evaluated for its anti-inflammatory potency by means of the carrageenan-induced rat paw edema assay . The assay was run on male Wistar rats (150 Â± 5g) purchased from the Institute of Biotechnology of Bahauddin Zakariya University (Multan, Pakistan). These rats were randomly divided into five groups with three rats in each group. These rats were allowed free access to food and water. The protocol involved injecting a 0.1 mL volume of 1% w/v carrageenan suspension in Normal saline into the sub-plantar tissue of each animal's right hind paw. This was immediately followed by applying to the injection site 1mL of the DDA-containing lotion over a 2 cm2 area. The control group was administered with lotion without enhancer. After 3 h, the extent of tissue inflammation was quantified by simply measuring the linear paw circumference .
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  on male Wistar rats (â‰¤ 450 g weight). To this end, a 1 mL aliquot of test formulation was applied to each animal's abdomen. The animal was placed in a dedicated cloth restrainer that was specially designed for this version of the flick test . At 30, 45 and 60 min after lotion administration, the animal's tail (2-5cm long) was immersed in water maintained at 53 Â± 1 Â°C. The reaction time was the time taken for the rat to flick its tail. In practice, the first reading was ignored and the reaction time was taken as the mean of the subsequent two readings. Each analgesic formulation was tested on 3 rats of each group.
Lastly, each formulation was assessed for irritancy by conducting modified Draize skin irritation tests  on male White New Zealand rabbits (3-4 kg) obtained from Novartis (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. A 0.5mL aliquot of each test lotion was applied to these areas, which were then covered with a plastic patch. After 4 h, the patch was removed and the 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.
Sensatory perception test
Sensatory perception test involved eleven untrained Caucasian volunteers, both male and female, ranging between 20 to 24 years in age. This study was ethically approved by the Human Volunteers Ethics Committee of Bahauddin Zakariya University (Pakistan). A small amount of test formulation was applied to a 12 cm2 area on the back of each volunteer's hand and left on for 10 min. Each volunteer rated the test lotion's 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 bad to excellent respectively. In addition, skin treatment sites were visually examined for signs of cutaneous irritancy. A confidence level of 95% was taken as significant.
Results and Discussion
In vitro characterization
All the lotion formulations were clear, transparent and homogeneous solutions upon preparation which exhibited a pH of 6.3 with no significant difference among all the formulated lotions. However, increasing PG and TO content in the formulated lotions decreased the viscosity from 89 Ã- 10-4 dyn.s.cm-2 for L1; 83 Ã- 10-4 dyn.s.cm-2 for L2; 78 Ã- 10-4 dyn.s.cm-2 for L3 and 71 Ã- 10-4 dyn.s.cm-2 for L4. A similar to viscosity trend was observed in the case of spreadability of formulated lotions where spreadability was decreased upon subsequent increase in the PG and TO content i.e. 3.02 Â± 0.12 mg.cm.s-1 for L1, 2.14 Â± 0.17 mg.cm.s-1 for L2, 2.12 Â± 0.21 mg.cm.s-1 for L3 and 2.01 Â± 0.09 mg.cm.s-1 for L4. Statistical analysis revealed that there was a significant difference between L1 and L4 spreadability. Overall, an increase in PG and TO content in the lotion formulation decreased the viscosity and spreadability.
During the three month accelerated stability testing, none of the formulation showed any changes to the appearance, color and transparency. Furthermore, there was an insignificant difference among all the formulated lotions in terms of pH, viscosity, spreadability and drug content over the course of accelerated stability testing period suggesting the formulated lotions were fairly stable.
In vitro permeation studies
In vitro permeation profile is an important tool that predicts how drug will behave in vivo. In vitro permeation of DDA containing lotions were performed using two model membranes, namely polydimethylsiloxane and rabbit skin. As far as we could ascertain, there is no published data for DDA permeation using rabbit and polydimethylsiloxane membrane model. Figure 1 displays the cumulative amount of DDA permeated through polydimethylsiloxane membrane as a function of time. The steady-state flux was determined from the slope of the linear portion of the cumulative amount of drug permeation versus time plot. Permeability coefficients were calculated by applying Fick's laws of diffusion. Flux enhancement ratio (ER) was calculated from the proportion of flux in the presence and absence of enhancer in the lotion formulation.
Figure 1: Cumulative drug permeated through polydimethylsiloxane membrane (n=5)
It should be noted that the initial burst in the drug permeation exhibited a non-ideal behaviour. This effect was attributed to the polydimethylsiloxane membrane material undergoing perturbation due to interaction between polydimethylsiloxane membrane and vehicle system, consequently, increasing the diffusion coefficient of the drug. Therefore, it was decided to select period of 15 to 180 minute in order to calculate the steady-state flux. The cumulative amount of drug permeated as a function of time revealed that increasing enhancer concentration in the lotion markedly increased the permeation of DDA as compared to that of the control. Moreover, there was no significant difference observed in permeation of DDA between all the formulated lotions suggesting a concentration independent increase in the permeability of DDA in case of polydimethylsiloxane membrane. The flux and permeability coefficient values were significantly different from that of the control. Furthermore, a gradual increase in the flux rates and permeability coefficient values was observed with increasing concentration of PG and TO. Lag time (tlag) is the time taken by the drug to reach its steady-state and data revealed that L4 has the lowest tlag and DDA permeation has reached to its steady-state quicker than the other formulations containing lower or no enhancer content. The permeation profile and flux enhancement ratio are summarized in Table II.
Table II: Permeation profile of DDA across polydimethylsiloxane membrane
Lag time (tlag)
(10-4x Kp) (cm/min)
0.038 Â± 0.006
47.23 Â± 9.98
0.95 Â± 0.14
0.863 Â± 0.02
60.54 Â± 2.00
21.58 Â± 0.58
0.946 Â± 0.02
51.83 Â± 2.38
23.65 Â± 0.49
1.013 Â± 0.009
50.84 Â± 1.22
25.32 Â± 0.22
1.196 Â± 0.02
42.09 Â± 2.37
29.91 Â± 0.48
Results are presented as mean Â± SD (n = 5).
Fluxes and permeability coefficients were measured for all the DDA containing lotions across rabbit skin and cumulative amount of DDA permeated across rabbit skin as a function of time is shown in Figure 2. The drug permeation was more or less linear till 700 minutes of the study after that it reached to the steady-state region where drug permeation rate was constant over the time period from 700 to 1440 minutes. Therefore, the time period after 700 minutes was deliberately ignored in order to calculate the steady-state flux.
Figure 2: Cumulative drug permeated through rabbit skin (n=5)
It was noteworthy that the permeation rate was ceased after approximately ~700 minutes which could be attributed to the precipitation of DDA on the surface of rabbit skin which reduced the effective diffusion area, consequently, sinking the permeation of DDA. There was a gradual increase in flux rate with increasing content of PG and TO in the lotions while a remarkable improvement in the permeability coefficient was observed for all lotion formulations in comparison to that of control. Statistical analysis revealed a significant difference (P<0.005) in permeability coefficients as compared to control for all the formulated lotions. The permeation data of DDA across rabbit skin is shown in the Table III. The enhancement ratio on the basis of flux was highest for the L4 (4.7 folds) and lowest for the L1 (3.0 folds) which was related to the enhancer concentration in DDA containing lotions. It was interesting to notice that the lag time increased with the increase in enhancer concentration which might be attributed to the impact of enhancer on the apparent permeability of the DDA. The contrasting lag times for DDA permeation through polydimethylsiloxane membrane and rabbit skin could be due to the structural differences between both membranes and how the permeation enhancer interacts with the membrane. It can be explained by the fact that TO can penetrate rapidly and deposit in the skin owing to its physicochemical properties, thus, causing a delayed permeation which consequently enhanced lag times with higher concentrations in the case of DDA permeation across rabbit skin.
Table III: Permeation profile of DDA across rabbit skin
Lag time (tlag)
(10-8x Kp) (cm/min)
0.142 Â± 0.001
37.53 Â± 1.81
0.053 Â± 0.003
0.428 Â± 1.02
19.28 Â± 0.67
20.34 Â± 0.05
0.548 Â± 2.913
67.31 Â± 2.60
26.07 Â± 0.014
0.621 Â± 1.86
97.01 Â± 2.30
29.85 Â± 0.009
0.668 Â± 3.13
142.73 Â± 1.10
31.78 Â± 0.015
Results are presented as mean Â± SD (n = 5).
In vivo studies data
The graph in Figure 3 shows the data obtained from the carrageenan challenge anti-inflammatory tests. It can be seen that application of each of the DDA-containing formulations significantly reduced tissue inflammation in the rat model. In contrast, application of LC did not significantly affect inflammation because of its low permeation into the skin. Another noteworthy point from statistical analysis is that while anti-inflammatory effect of L1 was significantly different than L2, L3 and L4, the latter three formulations did not differ significantly from each other in anti-inflammatory potency which is explainable on the basis of permeability coefficient which was insignificantly different for L2, L3 and L4.
Figure 3: Bar graphs showing the in vivo edema reduction induced by each DDA formulation in carrageenan-challenged rabbits. Error bars represent SD values, with n=3.
Figure 4 displays the data derived from the hot tail antinociception studies. The graph clearly indicates that the reaction time measured following treatment with a DDA-containing lotion was always significantly longer than the reaction time measured following treatment with the LC. Furthermore, the extent of induced antinociception followed the trend; L4 > L3 > L2 > L1, indicating that PG and TO content influenced antinociception potency of DDA by enhancing its permeation.
Figure 4: Bar graph diagram showing the in vivo tail flick response times (antinociception) associated with each DDA formulation at 30, 45 and 60 mins after lotion application. Error bars represent SD values, with n=3.
With respect to the Draize irritation tests, results indicated that application of all lotion formulation were invariably associated with no skin irritation throughout the entire 14 day period. With respect to the L4 formulation, all tested rabbits showed some mild erythema (score of 1) by day 14 although not at the earlier observation times (data not shown).
Sensatory perception data
The volunteers rated all the DDA containing lotions 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 (Data not shown).
Based on the results from this study, it was possible to conclude that PG and TO has effectively improved the permeability of DDA. Although for all the formulations studied the best effective in vitro permeation and in vivo performance was achieved when the highest PG and TO concentration was used in the formulation, L4 in this case.
The authors would like to thank Bahauddin Zakariya University (Multan, Pakistan) for providing financial support for this research.