Development of topical gel containing aceclofenac ...

chemical engineering research and design 9 2 ( 2 0 1 4 ) 2095–2105

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Development of topical gel containing aceclofenac-crospovidone solid dispersion by “Quality by Design (QbD)” approach Sougata Jana a , Syed Ansar Ali b , Amit Kumar Nayak c,∗ , Kalyan Kumar Sen a , Sanat Kumar Basu d a

Department of Pharmaceutics, Gupta College of Technological Sciences, Ashram More, Asansol 713301, W.B., India Department of Pharmaceutics, Karnataka College of Pharmacy, 33/2 Thirumenahalli, Bangalore 560064, India c Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj 757086, Odisha, India d Division of Pharmaceutics, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, W.B., India b

a b s t r a c t This article describes the development, optimization, and evaluation of Carbopol 940 topical gel containing aceclofenac-crospovidone (1:4) solid dispersion using “Quality by Design (QbD)” approach based on 23 factorial design. The effect of crospovidone, tri-ethanolamine, and ethyl alcohol amount on the drug permeation profile of the topical gel containing aceclofenac-crospovidone solid dispersion was optimized by 23 factorial design. The optimized gel showed improved permeation profile with cumulative drug permeation of 26.262 ± 2.157%, and permeation flux of 0.059 ± 0.011 ␮g/cm2 /h. These gels were characterized by pH, viscosity, gel strength and FTIR study. The in vivo antiinflammatory activity of the optimized gel was evaluated in rats using carrageenan-induced rat-paw oedema model and found excellent anti-inflammatory comparable with a marketed gel without producing any skin irritation. © 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Topical gel; Carbopol 940; Aceclofenac; Factorial design; QbD; Optimization

1.

Introduction

In general, gels are formed from a liquid phase that has been thickened with other components. Among them, Carbopol gels offer good alternatives to oil based topical formulations. Carbopols are polymers of acrylic acid cross-linked with polyalkenyl ethers or divinyl glycol. They are readily hydrated to swell. Because of the hydrophilic nature, the cross-linked structures of Carbopols make them potential candidates for use as gel type formulation for topical use (Carnali and Naser, 1992; Garcia-Gonzalez et al., 1994). Topical use of these gels is advantageous, as they possess good rheological properties resulting in long residue times at the site of administration. Due to their extremely high molecular weight, they cannot penetrate the skin and offer good alternatives to oil based ointment formulations.

∗

Aceclofenac, chemically [2-(2 ,6 -dichlorophenyl)amino] phenylacetoxyacetic acid, is used as a non-steroidal antiinflammatory drug (NSAID) indicated for the symptomatic treatment of pain and inflammation (Chakraborty et al., 2010). It is also used in the treatment of arthritis, osteoarthritis, rheumatoid arthritis and ankylosing spondylitis (Yadav et al., 2010). However, like other NSAIDs, oral administration of aceclofenac is also associated with gastrointestinal side effects like gastric ulceration, gastrointestinal bleeding and liver and kidney trouble (Insel, 1992). In view of adverse drug reaction associated with oral formulations, aceclofenac is increasingly administered by topical route (Heyneman et al., 2000). Furthermore, the topical route of administration eliminates side effects, increases patient compliance, avoids first-pass metabolism, and maintains the plasma drug level for a longer period. Aceclofenac has a poor aqueous solubility (Nagariya

Corresponding author. Tel.: +91 9583131603. E-mail address: [email protected] (A.K. Nayak).

Available online 4 February 2014 0263-8762/$ – see front matter © 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cherd.2014.01.025

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et al., 2010) that may cause a problem of migrating through hydrophilic base. Crospovidone is a synthetic polymer derived from the monomer of vinyl pyrrolidone by popcorn polymerization (Haaf et al., 1985). It is mainly used as disintegrant in tablets and capsules. Crospovidone has been used as a carrier of solid dispersion for various drugs to improve their aqueous solubility (Loganathan et al., 2000; Makiko et al., 2005). In the present study, an attempt was made to develop Carbopol 940 gel for topical application containing solid dispersion of aceclofenac using crospovidone as carrier to improve the skin permeation profile of aceclofenac using “Quality by Design (QbD)” approach. QbD approach encompasses designing and developing formulations, in which manufacturing processes ensure predefined product specifications (Nayak et al., 2011). The important part of this approach is to understand how process and formulation parameters affect the product quality and subsequent optimization parameters with respect to final specifications (Maltesen et al., 2008). QbD refers to the achievement of certain predictable quality with desired and predetermined specifications. Therefore, a very useful component of the QbD is the understanding of various factors (variables) and their interactions by a desired set of experiments using a statistical tool (Menini et al., 2012). In the present investigation, the planned aceclofenac-loaded Carbopol 940 topical gel containing aceclofenac-crospovidone solid dispersion was optimized in terms of cumulative drug permeation through the excised mouse skin after 10 h (%) and permeation flux (␮g/cm2 /h) by three-factor and two-level (23 ) factorial design. The considered factors were amount of crospovidone (mg), amount of tri-ethanolamine (ml) and amount of ethanol (ml). The selected QbD strategy allowed an efficient selection of the best formulation composition and of the most suitable experimental conditions in the shortest time and with the minimum number of experiments. The best formulation was studied for in-vivo pharmacodynamic performance in carrageen-induced rat paw oedema model and was compared with marketed gel formulation.

2.3. Characterization of aceclofenac-crospovidone solid dispersion 2.3.1.

Saturation solubility measurement

The known excess samples (solid dispersions, physical mixture and aceclofenac) of 10 mg equivalent weight of aceclofenac was added to 10 ml of phosphate buffer saline, pH 7.4 and these samples were rotated at 20 rpm in a water bath (37 ± 0.5 ◦ C) for 48 h. The samples were then filtered, suitably diluted, and analyzed by UV–vis spectrophotometer (Thermo Spectronic UV-1, USA) at 274 nm wavelength using appropriate blank solution.

2.3.2.

Differential scanning calorimetric (DSC) analysis

DSC analyses of the pure aceclofenac, and aceclofenaccrospovidone (1:4) solid dispersion were performed. The samples were heated to remove the moisture. Then the samples (7 mg) were placed into a platinum crucible 40␮l aluminium pan in hermetically sealed condition, where ∝-alumina powder was used as a reference. Thermograms were recorded from 30 ◦ C to 415 ◦ C at the heating rate of 10 ◦ C/min under a constant flow of an inert nitrogen gas atmosphere with the flow rate of 20 ml/min. These analyses were done using a differential scanning calorimeter (Perkin Elmer® Instrument, Pyris diamond, Osaka, Japan).

2.4. Preparation of Carbopol 940 gel containing aceclofenac-crospovidone solid dispersion Carbopol 940 gels containing aceclofenac-crospovidone solid dispersion were prepared according to the literature with little modification (Dua et al., 2010). In brief for each formulation, required amount of aceclofenac-crospovidone solid dispersion equivalent to 150 mg aceclofenac was dissolved in ethanol and deionised water, respectively. Both the solutions are mixed together thoroughly. Then 100 mg of Carbopol 940, previously soaked in 6.50 ml of deionised water overnight, was added to the above mixture with stirring at 500 rpm by magnetic stirrer (Remi Motors, India) for 1 h. Finally, weighed quantity of tri-ethanolamine was added to obtain a clear gel.

2.

Materials and methods

2.5.

2.1.

Materials

For the optimization of Carbopol 940 gels containing aceclofenac-crospovidone solid dispersion, a 23 factorial design was employed. Amount of crospovidone (X1 , mg), amount of tri-ethanolamine (X2 , ml) and amount of ethanol (X3 , ml) were selected as independent variables (factors), which were varied at two levels (low and high). The cumulative drug permeation through the excised mouse skin after 10 h (CDP10 , %) and permeation flux (PF, ␮g/cm2 /h) were used as dependent variables (responses). Design-Expert 8.0.6.1 software (Stat-Ease Inc., USA) was used for generation and evaluation of the statistical experimental design. The matrix of the design including investigated factors and responses are shown in Table 2. For optimization, effects of various independent variables upon measured responses were modelled using following mathematical model equation involving independent variables and their interactions for various measured responses generated by 23 factorial design is as follows:

Aceclofenac was obtained as the gift sample from Suyash Lab, India. Carbopol 940 and crospovidone were obtained as gift sample from C.I. Laboratories, India. Tri-ethanolamine and ethanol were commercially purchased from Merck, India and Bengal Chemical & Pharmaceuticals Ltd., India. All other reagents were of analytical grade and commercially available.

2.2. Preparation of aceclofenac-crospovidone solid dispersion Aceclofenac-crospovidone (1:4) solid dispersion was prepared by solvent evaporation technique. Aceclofenac was dissolved in ethanol to get clear solution. Then, crospovidone was dispersed as fine particles and the solvent was removed by evaporation on a water bath at 60 ◦ C. The dried mass was stored in desiccator until constant mass was obtained, pulverized and passed through sieve no. 22.

Experimental design

Y = b0 + b1 X1 + b2 X2 + b3 X3 + b4 X1 X2 + b5 X1 X3 + b6 X2 X3 where Y is the dependent variable, while b0 is the intercept, b1 , b2 , b3 , b4 , b5 , and b6 are regression coefficients; X1 , X2 and X3


are undependable variables; X1 X2 , X2 X3 , and X1 X3 are interactions between variables. One-way ANOVA was applied to estimate the significance of the model (p < 0.05) and individual response parameters.

2.6. Characterization of Carbopol 940 gel containing aceclofenac-crospovidone solid dispersion 2.6.1.

pH determination

pH of the prepared gel was measured using a digital pH meter (Systronics Instruments, India) by placing the glass electrode completely into the gel system and compared with marketed formulation.

2.6.2.

Determination of drug content

For each batch, 500 mg of gel was dissolved in 500 ml of phosphate buffer saline, pH 7.4 with stirring at 500 rpm by magnetic stirrer (Remi Motors, India) for 1 h. After suitable dilution, the absorbance of the above solution was analyzed by UV–vis spectrophotometer (Thermo Spectronic UV-1, USA) at 274 nm wavelength using appropriate blank solution.

2.6.3.

Measurement of gel strength

The gel strength of formulated gels were determined after 48 h of preparation by measuring the weight required to move upper plate by 3 cm, when 1 g of each gel was placed between two 20 cm × 20 cm plates. The gel strength was calculated by using the formula: S=

M×L T

where S is the gel strength, M is the weight tied to the upper slide, L is the length glass slide travelled and T is time taken. Homogeneity of various gel formulations were tested by visual observations (Covert, 1986).

2.6.4.

Viscosity measurement

The viscosity of the formulations were determined by using a Brookfield DV III ultra V6.0 RV cone and plate viscometer (Brookfield Engineering Laboratories, Middleborough, MA) at 25 ± 0.3 ◦ C; the software used for calculation was Rheocalc V2.6 (Chawla and Saraf, 2012).

Institutional Animal Ethical Committee, under registration number 955/A/06/CPCSEA. The animals were sacrificed using anaesthetic ether. The hair of abdominal skin was removed by using an animal hair clipper. A full thickness of skin was taken out and the fat adhering to the dermis side was removed by using surgical scalpel. Finally, the skin was rinsed using phosphate buffer, pH 7.4 and packed in aluminium foil. The skin sample was stored at −20 ◦ C and was used within 24 h. The ex vivo permeation through excised mouse skin was performed by Franz diffusion cells. The cells consist of two chambers, the donor and the receptor compartment with an available diffusion area of 0.949 cm2 . The donor compartment was open at the top and was exposed to atmosphere. The excised mouse skin was mounted between the compartments of the diffusion cell with stratum corneum facing the donor compartment and clamped into position. Magnetic stirrer bars were added to the receptor chambers and filled with the receptor phase. Phosphate buffer saline, pH 7.4 was used as the receptor medium. The small concentration of sodium azide (0.0025%, w/v) was added to prevent any microbial growth (Pillai and Panchagnula, 2004). The entire setup was placed over magnetic stirrer, and the temperature was maintained at 37 ± 0.5 ◦ C. The skin sections were initially left in the Franz cells for 2 h in order to facilitate hydration of the skin samples. 1 g of each gel formulation was applied onto the excised mouse skin fitted on the Franz diffusion cell. 1 ml of medium was collected from the receptor compartment at predetermined intervals over study period and replaced with the same amount of fresh buffer. The amount of permeated drug was analyzed by UV–vis spectrophotometer (Thermo Spectronic UV-1, USA) at 274 nm wavelength using appropriate blank solution.

2.9.

FTIR spectroscopy of pure aceclofenac, physical mixture of aceclofenac-crospovidone (1:4) solid dispersion and optimized aceclofenac gel were performed. Each sample were ground thoroughly with KBr (1:19) and then pellets were prepared using a hydraulic press under a hydraulic pressure of 100 kg/cm2 for 10 min. These KBr pellets containing samples were analyzed by using a FTIR spectroscope (Spectrum BX, Perkin-Elmer® Instruments, USA). The pellets were placed one by one in the sample holder. Spectral scanning was taken in wavelength region between 4000 and 400 cm−1 at a resolution of 4 cm−1 with 2 mm/s scan speed.

2.8.

Ex vivo permeation study

The ex vivo permeation of aceclofenac from Carbopol 940 gels containing aceclofenac-crospovidone solid dispersion were performed using excised skin of Swiss albino mice (weight 162–188 g). The experiment was approved by

Permeation data analysis

The amount of aceclofenac permeated from various gels through excised mouse skin was plotted against the function of time. The slope and intercept of the linear portion of plots were derived by regression. The permeation fluxes for each gel were calculated as the slope divided by the skin surface area (Malakar et al., 2011; Nayak et al., 2010): Jss =

2.7. Fourier transform-infrared (FTIR) spectral analysis

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(dQ/dt)ss × 1 , A

where Jss is the steady-state permeation flux (␮g/cm2 /h), A is the area of skin tissue (cm2 ) through which drug permeation takes place, and (dQ/dt)ss is the amount of drug passing through the skin per unit time at a steady state (␮g/h).

2.10.

In vivo evaluation

The experiment was approved by Institutional Animal Ethical Committee, under registration number 955/A/06/CPCSEA. The carrageenan-induced rat-paw oedema model (Winter et al., 1962) was performed to assess anti-inflammatory activity evaluation of the optimized gel. Male Sprague Dawley rats weighing 200–250 g were used for the experiment. The acclimatized rats were kept fasting for 24 h with water ad libitum. The anti-inflammatory effect of the optimized aceclofenac gel was evaluated by applying 1 g of optimized formulated gel on the skin (1 cm2 ) back of the rats. The control group was treated with the normal saline. After 3 h interval, 0.1 ml of a 1% carrageenan solution in physiological saline as the control was injected intradermally in the right hind paw of the

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rat. The oedema volumes are measured using plethysmometer (Ugo Bacile, model 7150) after 3 h of carrageenan injection. The extent of swelling (%) were calculated from the difference in the volume between immediately and 3 h after the carrageenan injection (6 rats in each group) using the following formula (Shin et al., 2000): Swelling (%) =

V − V1 × 100 V1

where V is the volume 3 h after the carrageenan injection in the sole of the foot and V1 is the volume immediately after the injection.

2.11.

Skin irritation test

The skin irritation was performed on healthy New Zealand rabbits (2.07–2.56 kg). The experiment was approved by Institutional Animal Ethical Committee, under registration number 955/A/06/CPCSEA. For each gel, five rabbits were selected and after cleaning, the dorsal skin 1 g of optimized gel was applied on an area of 2 in.2 to the back. The animals were kept under standard laboratory conditions at 25 ± 1 ◦ C and relative humidity of 55 ± 5%. The animals were housed with free access to a standard laboratory diet and water ad libitum. The rabbits were then observed for lesions or irritation in presence and absence of sunlight exposure of 30 min (Van-Abbe et al., 1975).

2.12.

Statistical analysis

Statistical optimization was performed using Design-Expert 8.0.6.1 software (Stat-Ease Inc., USA). The in vivo data were tested for significant differences (p < 0.05) by paired samples ttest. All other data were analyzed by average mean ± standard deviation. The simple statistical analysis and paired samples t-test were conducted using MedCalc software version 11.6.1.0.

3.

physical mixture (0.126 ± 0.012 mg/ml) and pure aceclofenac (0.091 ± 0.009 mg/ml). This might be attributed to an improved wetting of drug particles and localized solubilization by the hydrophilic polymeric carrier, crospovidone.

3.1.2.

DSC analysis

DSC thermograms of pure drug aceclofenac and prepared aceclofenac-crospovidone (1:4) solid dispersion are presented in Fig. 1. Thermogram of pure aceclofenac (a) showed sharp peaks at 152 ◦ C and 275.60 ◦ C, indicating the melting point and polymorphic nature of aceclofenac, respectively. However, the thermogram of prepared aceclofenac-crospovidone (1:4) solid dispersion depicted that there was a noticeable reduction in endothermic peak heights and heat of fusion compared to pure aceclofenac, suggesting the change of crystalline state in pure aceclofenac to amorphous form in aceclofenac solid dispersion. It has been understood that transforming the physical state of the drug to amorphous or partially amorphous state leads to a high-energy state, resulting in enhanced aqueous solubility and faster dissolution of drug candidates.

Results and discussions

3.1. Characterization of aceclofenac-crospovidone solid dispersion 3.1.1.

Saturation solubility

Aceclofenac-crospovidone (1:4) solid dispersion was prepared by solvent evaporation technique to improve the solubility of aceclofenac. The concentration of saturated aqueous solution of prepared aceclofenac-crospovidone (1:4) solid dispersion was measured and compared with the corresponding physical mixture, and pure aceclofenac (Table 1). The prepared solid dispersion showed improved aqueous solubility of aceclofenac (0.248 ± 0.020 mg/ml) than that of corresponding Table 1 – Saturation solubility of pure aceclofenac and prepared aceclofenac-crospovidone (1:4) solid dispersion. Samples Pure aceclofenac Physical mixture of aceclofenac-crospovidone (1:4) Aceclofenac-crospovidone (1:4) solid dispersion a

Fig. 1 – DSC thermogram of aceclofenac and aceclofenac-crospovidone (1:4) solid dispersion.

Mean ± S.D., n = 3.

Saturation solubility (mg/ml)a 0.091 ± 0.009 0.146 ± 0.012 0.248 ± 0.020

3.2. Optimization of Carbopol 940 gel containing aceclofenac-crospovidone solid dispersion 23 (three-factors, two-levels) factorial design was employed for the optimization of Carbopol 940 gels containing aceclofenaccrospovidone (1:4) solid dispersion. A total 8 trial formulations were proposed by the 23 factorial design for three independent variables (factors) namely, amount of crospovidone (X1 , mg), amount of tri-ethanolamine (X2 , ml) and amount of ethanol (X3 , ml) were selected as independent variables (factors), which were varied at two levels (low and high). The effects of these independent variables on the CDP10 (%) and PF (␮g/cm2 /h) were investigated as responses and optimized. According to experimental design, 8 trial formulations were formulated and evaluated for their responses investigated. The matrix of the design including investigated factors and responses are shown in Table 2. Design-Expert 8.0.6.1 software (Stat-Ease Inc., USA) was used for generation and evaluation of the statistical experimental design. The values of responses for each trial formulations were fitted in the design to get model equations for each response. The model equation relating CDP10 (%) as response became: CDP10 (%) = 4.359 + 0.031X1 − 10.508X2 + 1.783X3 + 0.183X1 X2 + 4.811 × 10−3 X1 X3 − 7.271X2 X3 [R2 = 0.9996; F-value = 2596.78; p < 0.05].

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Table 2 – 23 full factorial design (coded values in bracket) with observed response values for different Carbopol 940 gels containing aceclofenac-crospovidone (1:4) solid dispersion. Code

Normalized levels of independent variables (factors) employed Crospovidone (mg), X1

F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8

Tri-ethanolamine (ml), X2

150.00 (+1) 150.00 (+1) 150.00 (+1) 150.00 (+1) 25.00 (−1) 25.00 (−1) 25.00 (−1) 25.00 (−1)

Ethyl alcohol (ml), X3

0.10 (+1) 0.10 (+1) 0.02 (−1) 0.02 (−1) 0.10 (+1) 0.10 (+1) 0.02 (−1) 0.02 (−1)

4.00 (+1) 1.00 (−1) 4.00 (+1) 1.00 (−1) 4.00 (+1) 1.00 (−1) 4.00 (+1) 1.00 (−1)

CDP10 (%)a,c 17.808 12.546 18.845 11.697 9.286 5.687 12.016 6.813

± ± ± ± ± ± ± ±

0.710 1.500 1.137 0.527 0.466 1.026 0.404 0.215

PF (␮g/cm2 /h)b,c 0.040 0.026 0.041 0.025 0.022 0.014 0.025 0.016

± ± ± ± ± ± ± ±

0.002 0.004 0.003 0.003 0.005 0.002 0.004 0.002

(+1) = higher values and (−1) = lower values. a b c

Cumulative drug permeation after 10 h (%). Permeation flux. Mean ± S.D., n = 3.

The model equation relating PF (␮g/cm2 /h) as response became: PF (␮g/cm2 /h) = 0.012 + 5.167 × 10−5 X1 − 0.022X2 + 2.275 × 10−3 X3 + 2.500 × 10−4 X1 X2 + 1.733 × 10−5 X1 X3 − 6.250 × 10−3 X2 X3 [R2 = 0.9998; F-value = 910.33; p < 0.05]. The results of ANOVA, as shown in Table 3, indicated that all models were significant (p < 0.05) for all response parameters investigated. Model simplification was carried out by eliminating non-significant terms (p > 0.05) in model equations (Nayak et al., 2011), giving: CDP10 (%) = 4.359 + 0.031X1 − 10.508X2 + 1.783X3 + 0.183X1 X2 + 4.811 × PF (␮g/cm2 /h) = 0.012 + 5.167 × 10−5 X1 + 2.275 × 10−3 X1 X3 ; 10−3 X3 + 1.733 × 10−5 X1 X3 . Each response coefficient was studied for its statistical significance by Pareto charts as shown in Fig. 2 and Fig. 3. These charts depicted the statistical significance of each response coefficient. Coefficients with t values of effects above the Bonferroni line are designated as significant coefficient; coefficients with t values of effects between Bonferroni line and t limit line are termed as coefficients likely to be significant, while coefficients with t values of effects below the t limit line is statistically insignificant coefficient (Shah and

Pathak, 2010). Therefore, these Pareto charts supported also the ANOVA results for the model simplification by eliminating non-significant terms (p > 0.05) in both the model equations. In addition, Design-Expert 8.0.6.1 software generated threedimensional response surface plots (Fig. 4 and Fig. 5) and corresponding contour plots (Fig. 6 and Fig. 7) to estimate the effects of the independent variables (factors) on each response investigated (CDP10 , % and PF, ␮g/cm2 /h). The threedimensional response surface plot is very useful in learning about the main and interaction effects of the independent variables (Nayak and Pal, 2011; Malakar et al., 2012). The three-dimensional response surface plots relating CDP10 (%) (Fig. 4a–c) depict the increase in CDP10 with the increasing of both the amount of crospovidone (X1 ), and ethyl alcohol (X3 ), whereas slight decreasing of CDP10 was found with the increment of the amount of tri-ethanolamine (X2 ). On the other hand, the three-dimensional response surface plots relating PF (␮g/cm2 /h) (Fig. 5a–c) also indicate the increase in PF with the increasing of both the amount of crospovidone (X1 ), and ethyl alcohol (X3 ), whereas slight decreasing of PF was found with the increment of the amount of tri-ethanolamine

Table 3 – Summary of ANOVA for the response parameters. Source

Sum of squares

d.f.a

Mean square

F value

p-value Prob > F

b

(a) For CDP10 (%) Model X1 X2 X3 X1 X2 X1 X3 X2 X3

(b) For PF (␮g/cm2 /h)c Model X1 X2 X3 X1 X2 X1 X3 X2 X3

154.88 91.76 2.04 56.24 1.68 1.63 1.52 6.828 × 10−4 3.781 × 10−4 3.127 × 10−6 2.761 × 10−4 3.125 × 10−6 2.112 × 10−5 1.125 × 10−6

6 1 1 1 1 1 1

6 1 1 1 1 1 1

25.81 91.76 2.04 56.24 1.68 1.63 1.52 1.138 × 10−4 3.781 × 10−4 3.127 × 10−6 2.761 × 10−4 3.125 × 10−6 2.112 × 10−5 1.125 × 10−6

2596.78 9230.98 205.65 5658.03 169.18 163.69 153.16

0.0150 (S) 0.0066 (S) 0.0443 (S) 0.0083 (S) 0.0488 (S) 0.0497 (S) 0.0513 (NS)

910.33 3025.00 25.00 2209.00 25.00 169.00 9.00

0.0254 (S) 0.0116 (S) 0.1257 (NS) 0.0135 (S) 0.1257 (NS) 0.0489 (S) 0.2048 (NS)

X1 , X2 and X3 represent amount of crospovidone (mg), tri-ethanolamine (ml), and ethyl alcohol (ml), respectively; X1 X2 , X1 X3 , and X2 X3 are the interaction effects. S and NS indicate significant and not significant, respectively. a b c

Degree of freedom. Cumulative drug permeation after 10 h (%). Permeation flux.

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Fig. 2 – Pareto chart relating CDP10 (%). (X2 ). The two-dimensional contour plots (Fig. 6 and Fig. 7) were presented significant nonlinear relationships between the amount of crospovidone (X1 ) and tri-ethanolamine (X2 ) and amount of crospovidone (X1 ) and ethyl alcohol (X3 ) in case of CDP10 (%); whereas in case of PF (␮g/cm2 /h), the contour plots presented significant nonlinear relationship between amount of crospovidone (X1 ) and ethyl alcohol (X3 ) only. A numerical optimization technique using the desirability approach was employed to develop new formulations with desired response (desired quality). A constraint to maximizing the aceclofenac permeation was to set the goal to locate the optimum settings of independent variables for the optimized formula by QbD approach using the Design Expert 8.0.3 software based on the criterion of desirability. QbD approach stresses the need to understand the critical process parameters with the aim of achieving successful product development in predefined quality attributes thoroughly (Lionberger et al., 2008). Critical quality attributes are the properties that need to be controlled as they affect either patient safety or efficacy (Verma et al., 2009). To get the desired optimum responses, independent variables (factors)

were restricted to X1 = 260.00 mg, X2 = 0.01 ml, and X2 = 4.20 ml. In order to evaluate optimization capability of models generated according to the results of 23 factorial design, optimized Carbopol 940 gel containing aceclofenac-crospovidone (1:4) solid dispersion was prepared using the optimal process variable settings. The gel (F-O) was evaluated for various measured responses, i.e., CDP10 (%) and PF (␮g/cm2 /h). Table 4 lists the results of experiments with predicted responses by the mathematical model and those actually observed. The optimized gel (F-O) showed CDP10 of 26.262 ± 2.157%, and PF of 0.059 ± 0.011 ␮g/cm2 /h with small error-values (−4.139 and −3.279, respectively). This reveals that mathematical models obtained from the 23 factorial design were well fitted.

3.3. Characterization of Carbopol 940 gel containing aceclofenac-crospovidone solid dispersion 3.3.1.

pH

The pH of all formulated Carbopol 940 gels containing aceclofenac-crospovidone (1:4) solid dispersion were measured and found within the range of 6.52–7.24 (Table 5), which

Fig. 3 – Pareto chart relating PF (␮g/cm2 /h).


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Fig. 5 – Effect of crospovidone (mg), tri-ethanolamine (ml) and ethyl alcohol (ml) on PF (␮g/cm2 /h) presented by response surface plot (a–c).

Fig. 4 – Effect of crospovidone (mg), tri-ethanolamine (ml) and ethyl alcohol (ml) on CDP10 (%) presented by response surface plot (a–c). lies in the normal pH range of the skin. In the development of any topical formulation, the pH of the formulation is important. Because, the more acidic or more basic pH of the topical formulation can change the environment of the skin, which occasionally produce skin irritation upon application.

3.3.2.

0.407 ± 0.036 Pa-s. Among all formulations, the optimized gel exhibited maximum viscosity of 0.407 ± 0.036 Pa-s. From the viscosity result, it was clear that the viscosity of gels were increased with the increasing of crospovidone in their formula due to crospovidone hydrophilic nature. The gel strengths of these formulated gels were within the range between 0.116 ± 0.078 and 0.248 ± 0.065 g/cm/s. The gel strengths were found to be increased with increased viscosity value measured. The high gel strength might be related to the higher degree of polymer network in the gel.

Viscosity and gel strength

The viscosities of these formulated gels were determined by using a Brookfield DV-III ultra V6.0 RV at 25 ± 0.3 ◦ C; the software used for calculation was Rheocalc V2.6. The viscosity of these gels were within the range of 0.173 ± 0.017 to

3.3.3.

FTIR analysis

The FTIR spectra of aceclofenac, aceclofenac-crospovidone (1:4) solid dispersion, and optimized gel containing aceclofenac-crospovidone (1:4) solid dispersion are shown

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Fig. 6 – Effect of crospovidone (mg), tri-ethanolamine (ml) and ethyl alcohol (ml) on CDP10 (%) presented by contour plot (a–c).

in Fig. 8. The FTIR spectra of aceclofenac showed principal peaks at 3027.73 and 2936.75 cm−1 (due to both aromatic and aliphatic C H stretching vibrations, respectively), a band at 1717 cm−1 (due to C O stretching), a sharp band at 1771.97 cm−1 (due to C O stretching of carboxylic acid), a band at 3319.64 cm−1 (due to secondary N H rocking vibrations), and two sharp peaks at 716.11 cm−1 (due to 1,2-di-substituted C Cl stretching) (Mutalik et al., 2007a,b). The solid dispersion of aceclofenac-crospovidone (1:4) showed a sharp peak at 1256.86 cm−1 (due to C N stretching), which is a characteristic

Fig. 7 – Effect of crospovidone (mg), tri-ethanolamine (ml) and ethyl alcohol (ml) on PF (␮g/cm2 /h) presented by contour plot (a–c).

peak of crospovidone moiety, while the other bands present were similar due to the presence of pure drug. In case of optimized gel containing aceclofenac-crospovidone (1:4) solid dispersion, similar bands were observed as compared than that of previous one. Therefore, the FTIR analyses indicated absence of any significant interaction between the drug, aceclofenac and the excipients used.

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Table 4 – Results of experiments for confirming optimization capability. Code

Crospovidone (mg), X1

Tri-ethanolamine (ml), X2

Ethyl alcohol (ml), X3

Responses CDP10 (%)a,c

F-O

260.00

0.01

26.262 ± 2.157 27.396

4.20

−4.139

% Errord a b c d

PF (␮g/cm2 /h)b,c 0.059 ± 0.011 0.061 −3.279

Cumulative drug permeation after 10 h (%). Permeation flux. Mean ± S.D., n = 3. Percentage of error (%) = (actual value − predicted value)/predicted value × 100.

3.4.

Ex vivo permeation study

These gels were studied for ex vivo skin permeation through excised mouse skin. All these formulated gels containing aceclofenac-crospovidone (1:4) solid dispersion were sustained over 10 h, which was evidenced in ex vivo skin permeation study results (Fig. 9). In the optimization of these gels, it was found that drug permeation through mouse skin was found to be increased with increasing amount of crospovidone and ethyl alcohol. This phenomenon can be attributed by the aceclofenac solubility improvement with the increasing amount of crospovidone in the gels containing aceclofenac-crospovidone (1:4) solid dispersion and increased skin permeation enhancement capacity of increasing amount of ethanol, present in the gel formulations. The permeation fluxes for all these gels through the excised mouse skin were within the range between 0.014 ± 0.002 and 0.059 ± 0.011 ␮g/cm2 /h. Among all the formulated gels, the highest permeation profile (with the highest permeation flux of 0.059 ± 0.011 ␮g/cm2 /h) was observed in case of optimized gel (F-O), which contained 260.00 mg of crospovidone, 0.01 ml of tri-ethanolamine and 4.20 ml of ethyl alcohol.

3.5.

In vivo evaluation

The in vivo anti-inflammatory activity evaluation of the optimized topical gel was performed in male Sprague Dawley rats using carrageenan-induced rat-paw oedema model. The

Table 5 – Results of physical characterization of different Carbopol 940 gels containing aceclofenac-crospovidone (1:4) solid dispersion. Code

pH

F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-O

6.52 7.23 7.15 7.00 6.68 7.24 6.74 6.88 7.05

a

Viscosity (Pa-s)a 0.303 0.311 0.345 0.247 0.173 0.179 0.174 0.218 0.407

± ± ± ± ± ± ± ± ±

0.011 0.027 0.014 0.012 0.017 0.010 0.010 0.021 0.036

Gel strength (g/cm/s)a 0.187 0.177 0.172 0.158 0.144 0.144 0.124 0.116 0.248

± ± ± ± ± ± ± ± ±

0.042 0.051 0.091 0.078 0.022 0.058 0.078 0.078 0.065

Mean ± S.D., n = 3.

percent swelling (%) of rat paw oedema for control group, optimized gel and one marketed gel containing aceclofenac after 3 h were calculated and are presented in Fig. 10. However, a slightly lower percent of swelling (%) compared with control group was shown by the optimized gel and marketed gel. This could be well explained again by the fact that crospovidone network in Carbopol 940 topical gel containing aceclofenac-crospovidone (1:4) solid dispersion increased the rate of permeation of the drug, aceclofenac. This higher quantity of drug permeation resulted in increase in the intensity of response.

Fig. 8 – FTIR spectra of aceclofenac, aceclofenac-crospovidone (1:4) solid dispersion, and optimized Carbopol 940 gel containing aceclofenac-crospovidone (1:4) solid dispersion.

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Fig. 9 – Ex vivo permeation of aceclofenac from various Carbopol 940 topical gels containing aceclofenac-crospovidone (1:4) solid dispersion through excised mouse skin. with improved permeation profile and thus, improved patient compliance.

Conflict of interest The authors report no declarations of interest.

References Fig. 10 – A comparison of percent swelling in carrageenean-induced rats: control (a), optimized Carbopol 940 topical gels containing aceclofenac-crospovidone (1:4) solid dispersion (b) and a marketed aceclofenac gel (c).

3.6.

Skin irritation test

The development of erythema was monitored for 6 days and no significant development of any erythema or lesions on the surface of rabbit skin was found, which indicated the safety and acceptability of the optimized Carbopol 940 gel containing aceclofenac-crospovidone (1:4) solid dispersion for topical administration.

4.

Conclusion

Carbopol 940 topical gel containing aceclofenac-crospovidone solid dispersion was successfully developed by QbD approach based on 23 factorial design. These formulated gels showed sustained permeation of aceclofenac over 10 h in ex vivo skin permeation study using excised mouse skin. These gels were characterized by pH, viscosity, and gel strength. FTIR study clearly indicated absence of any significant interaction between the drug, aceclofenac and other excipients present in the formulation. The in vivo anti-inflammatory activity in male Sprague Dawley rats using carrageenan-induced ratpaw oedema model demonstrated that the optimized gel was comparable with a marketed gel without producing any skin irritation. Overall, these results indicated the promise of Carbopol 940 topical gel containing aceclofenac-crospovidone (1:4) solid dispersion for transdermal delivery of aceclofenac

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