Arch. Pharm. Res. DOI 10.1007/s12272-014-0367-8
RESEARCH ARTICLE
Mechanical properties, skin permeation and in vivo evaluations of dexibuprofen-loaded emulsion gel for topical delivery Sung Giu Jin • Abid Mehmood Yousaf • Mi Woon Son Sun Woo Jang • Dong Wuk Kim • Jong Oh Kim • Chul Soon Yong • Jeong Hoon Kim • Han-Gon Choi
•
Received: 16 January 2014 / Accepted: 9 March 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract The aim of this research was to evaluate the gel properties, skin permeation and in vivo drug efficacy of a novel dexibuprofen-loaded emulsion gel for topical delivery. In this study, the dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel were prepared with isopropanol, Tween 80, propylene glycol, isopropyl myristate and carbopol. Their mechanical properties such as hardness and adhesiveness were assessed. Moreover, their skin permeation, anti-inflammatory and anti-nociceptive efficacy were evaluated using Franz diffusion cell with the hairless mouse skin, the carrageenan-induced paw oedema test and paw pressure test in rat’s hind paws compared with the commercial hydrogel, respectively. The dexibuprofen emulsion gel and ibuprofen emulsion gel provided significantly higher hardness and adhesiveness than the commercial hydrogel. The dexibuprofen emulsion gel enhanced skin permeability by about twofold and 3.5-fold without lag time compared to the ibuprofen emulsion gel and the commercial hydrogel, respectively, suggesting its faster skin permeation. Moreover, the anti-inflammatory efficacy and alleviation in carrageenan-induced inflammation was in the order of dexibuprofen emulsion gel [ commercial hydrogel [ ibuprofen emulsion gel. The dexibuprofen S. G. Jin M. W. Son S. W. Jang J. H. Kim (&) Pharmaceutical Product Research Laboratories, Dong-A Pharm. Co. Ltd., Yongin-Si, Kyunggi-Do 449-905, South Korea e-mail: [email protected] S. G. Jin A. M. Yousaf D. W. Kim H.-G. Choi (&) College of Pharmacy, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-791, South Korea e-mail: [email protected] J. O. Kim C. S. Yong College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyongsan 712-749, South Korea
emulsion gel furnished significantly higher nociceptive thresholds than the ibuprofen emulsion gel and the commercial hydrogel, leading to the most improved anti-nociceptive efficacy. Thus, this dexibuprofen-loaded emulsion gel with good mechanical property, rapid skin permeation and excellent anti-inflammatory and anti-nociceptive efficacy would be a strong candidate for the topical delivery of anti-inflammatory dexibuprofen. Keywords Dexibuprofen Emulsion gel Hardness Adhesiveness Skin permeation Anti-inflammatory efficacy Anti-nociceptive efficacy
Introduction Topical non-steroidal anti-inflammatory drugs (NSAIDs) are usually applied as a gel or a patch on the skin in order to reach the deep subcutaneous tissues, show direct efficacy and avoid adverse effects (Baek et al. 2013; Rhee et al. 2013). These drugs inhibit cyclooxygenases and activate peroxisome proliferators-activated receptors, leading to reduced inflammation (Cox et al. 1999). Ibuprofen, an equal mixture of the R(-)-enantiomer and S(?)-enantiomer, has been widely used in many countries because it presents minimum side effects involving gastric damage seen for many NSAIDs. However, R(-)-ibuprofen undergoes ‘‘metabolic inversion’’ to the extent of 57–69 % to yield S(?)-ibuprofen. Dexibuprofen, S(?)-isomer ibuprofen, is pharmacologically more effective than ibuprofen; since the former reduced the level of gastric damage and improved the analgesic effects in comparison to the latter, this suggests that the oral and gel forms have excellent antiinflammatory effect (Bonabello et al. 2003; Cox et al. 1999; Kaehler et al. 2003).
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Recently, the gel product has been given the brand name NurofenÒ (Reckitt Benckiser GmbH; Mannheim, Germany) (Sabale and Vora 2012; Khan et al. 2011). In this study, in order to develop a novel dexibuprofen emulsion gel with better anti-inflammatory efficacy than ibuprofen emulsion gel, this gel was prepared with isopropanol, Tween 80, propylene glycol, isopropyl myristate and carbopol. The emulsion gel form provides more solubility and skin-permeability of poorly water-soluble drugs than the hydrogel form (Barot et al. 2012; Murdan 2005). Therefore, the emulsion gel form was developed for dexibuprofen. Its mechanical properties such as hardness and adhesiveness were assessed. Moreover, its skin permeation, anti-inflammatory and anti-nociceptive efficacy were evaluated using Franz diffusion cell with the hairless mouse skin, the carrageenan-induced paw oedema test and paw pressure test in rat’s hind paws and compared with results of the commercial hydrogel.
and isopropyl myristate at the weight ratio of 5:10:10:5, respectively. Carbopol (2.5 g) was also dissolved in 60.3 g of distilled water. Two solutions and diisopropanolamine (2.2 g) was entirely mixed and emulsified using a homogeniser (T-50 basic Ultra Turrax; IKA-Werke GmbH, Staufen, Germany), leading to the formation of 100 g emulsion gels. Mechanical properties of emulsion gel
Materials and methods
The mechanical properties of emulsion gel such as hardness and adhesiveness were investigated using a Texture Analyser (TA-XT2Ò Texture Analyzer; Microsystems, Haslemere, Surrey, UK). In this investigation, the hemispherical analytical probe (diameter 1 cm) was twice compressed into each sample at a defined rate (1 mm/s) and a trigger force of 0.1 N to a depth of 15 mm. From the resultant force–time plot (Fig. 1), the hardness and adhesiveness values were obtained from maximum force and the area of the negative region of curve, respectively (Bansal et al. 2009).
Materials
Skin permeation
Dexibuprofen and ibuprofen were supplied from Dong-A Pharm. Co. (Suwon, South Korea) and were of USP grade. Isopropanol, polysorbate 80 (Tween 80), propylene glycol, isopropyl myristate and diisopropanolamine were bought from the DC Chemical Co. (Seoul, South Korea). Carbopol (934P) was supplied from BF Goodrich (Brecsville, OH, USA). The commercial product (NurofenÒ; in hydrogel type, 5 % w/w ibuprofen) was purchased from Reckitt Benckiser GmbH (Mannheim, Germany). All other chemicals were of reagent grade and used without further purification.
All of the skin permeation experiments were conducted using Franz diffusion cells (FCDV-15, Labfine; Anyang, South Korea). The capacities of the donor and the receiver chambers were 1 and 5.5 mL, respectively. The surface area available for diffusion was 0.64 cm2. The receiver chamber temperature was maintained at 36.5 ± °C using a flow loop consisting of a water bath. The receptor chambers of the Franz diffusion cells were filled with phosphate buffer solution (pH 7.4) (5.5 ml) with continuous stirring using a magnetic stirrer. The hairless mouse skin was thawed at room temperature for a minimum of 2 h and was placed between the donor and receiver chambers with the stratum corneum facing the donor chamber. Then, three preparations, such as dexibuprofen-loaded emulsion gel, ibuprofen-loaded emulsion gel and the commercial hydrogel at a drug dose of 25 mg, were placed inside the donor chambers of Franz diffusion cells. At predetermined time intervals, 0.4 ml samples were withdrawn from the receptor chamber using a syringe. Fresh phosphate buffer solution (0.4 ml) was replaced in the receptor chambers. The collected samples were stored at 4 °C until further analysis. The concentrations of dexibuprofen (or ibuprofen) in the receptor chambers (10 ll) were measured using a HPLC system (Shimadzu, Japan) consisting of Class VP computer software, LC 10AD VP pump and SPD 10A VP UVVIS detector. The column was an Inertsil ODS-3 C18 column (5 lm, 150 9 4.6 mm). The mobile phase, a mixture of phosphate buffer (pH 3.5) and acetonitrile (4:6 v/v), was filtered through 0.45 lm membrane filter and
Animal Male Sprague–Dawley rats (7–9 weeks old, weighing 200–250 g) and hairless mice (8–10 weeks old) were acquired from the Charles River Company Korea (Orient, Seoul, Korea). All animal care and experimentations were done in accordance with the Guiding Principles in the Use of Animals in Toxicology, as accepted in 1989, reviewed in 1999, and modified in 2008 by the Society of Toxicology (SOT 2008). The criteria for the use of animals in research were also permitted by the Institutional Animal Care and Use Committees of Dong-A Pharmaceutical Corporation. Preparation of emulsion gel Dexibuprofen (5 g) or ibuprofen (5 g) was dissolved in a 30 g mixture of isopropanol, Tween 80, propylene glycol
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Mechanical properties, skin permeation Fig. 1 Mechanical force of dexibuprofen-loaded emulsion gels. Hardness and adhesiveness was obtained from maximum force and the area of the negative region of the curve, respectively
eluted at a flow rate of 1 ml/min. Effluents were monitored at 220 nm (ibuprofen, 214 nm) (Baek et al. 2012; Balakrishnan et al. 2009). In vivo efficacy tests The anti-inflammatory and anti-nociceptive efficacy of dexibuprofen-loaded emulsion gel were assessed by the carrageenan-induced paw oedema test and paw pressure test in rat hind paws, respectively (Bhandare et al. 2010; Kang et al. 2010). The suspension of 1 % carrageenan in saline (100 ll) was injected into the plantar side of the left hind paw of the rat, followed by percutaneous administration of three preparations such as dexibuprofen-loaded emulsion gel, ibuprofen-loaded emulsion gel and commercial product at the drug dose of 25 mg/kg after 1 h. Carrageenan caused visible redness and pronounced swelling that was well-developed 1 h after the injection. No treatment was used as a control. Paw volume was measured at 0, 2, 4, 7 and 10 h after percutaneous administration using a plethysmometer (Ugo Basile; Comerio, Italy). Furthermore, at 0, 2, 4, 7 and 10 h, the left hind paw of each rat was placed onto an analgesia meter (Ugo Basile; Comerio, Italy). Then, the weights were increased until the rat withdrew its paw. The nociceptive threshold (g), the force applied to the dorsal surface of the inflamed hind paw, was determined from the minimal weight (g) that caused each rat to withdraw its paw (Kang et al. 2010; Makuch et al. 2013).
Results and discussion Dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel were prepared by mixing and homogenising an oil phase and a water phase. For poorly water-soluble drugs, the emulsion gel form provided more solubility and permeability than the hydrogel form (Barot et al. 2012; Murdan 2005). Accordingly, the dexibuprofen emulsion
gel form was developed. In this study, isopropanol, Tween 80 and diisopropanolamine were used as an oil, surfactant and co-surfactant, respectively (Balakrishnan et al. 2009; Khan et al. 2011; Yerramsetty et al. 2010). In addition, carbopol was used as a thickening agent (Sheshala et al. 2013). In a preliminary study, carbopol provided stronger mechanical force to the emulsion gels compared to other hydrophilic polymers such as HPC, PVP and HPMC (data not shown). Then, their mechanical forces, such as hardness and adhesiveness, were investigated and compared with the commercial product. The commercial product (NurofenÒ) was a 5 % ibuprofen-loaded hydrogel prepared with carbopol as a base (Rhee et al. 2008; Serikov 2007). Hardness, which means the force required to attain a given deformation, was found from maximum force (Bansal et al. 2009). Furthermore, adhesiveness, which is the work required to overcome the attractive forces between the surface of the sample and the surface of the probe, was calculated from the area of the negative region of the curve (Fig. 1). Dexibuprofen-loaded emulsion gel and ibuprofenloaded emulsion gel gave significantly higher hardness (Fig. 2a) and adhesiveness (Fig. 2b) than the commercial hydrogel (243 ? 4 vs. 243 ? 5 vs. 227 ? 4 g; 1,999 ? 35 vs. 1,959 ? 36 vs. 1,810 ? 22 g s). However, there were no significant differences in the values between dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel. Thus, the emulsion gels prepared in this study had the excellent mechanical force compared to the commercial hydrogel due to the use of carbopol, a very highly viscous polymer (Sheshala et al. 2013). Moreover, the emulsion was more viscous than the solution. Therefore, the emulsion gel provided more viscosity than the general hydrogel with a single water phase (Murdan 2005). Next, the skin permeation study was performed using Franz diffusion cell with hairless mouse skin compared to the commercial hydrogel. From Fig. 3, the cumulative amounts of drug permeated (Mt, lg/cm2) were calculated as the total amount of drug permeated through the skin during a time period of 24 h (Gannu et al. 2010). Dexibuprofen-loaded emulsion gel and ibuprofen-loaded
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(A)
(B)
1000
500
0 6
12
18
24
Time (h) Fig. 3 Permeation profiles of drug in the emulsion gels. Each value represents the mean ± SD (n = 5)
emulsion gel showed significantly cumulative amounts of drug permeation compared to the commercial ibuprofenloaded hydrogel. The cumulative amounts of drug permeated from dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel were not significantly different. Table 1 shows their permeability parameters, such as permeability coefficient and lag time. The lag times (Lt) were
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el hyd rog
ge l em u ls io n fe n
ial
hy dr
gel fen
upr o De x ib
1500
Co mm erc
em u ls io n
mu lsio n ne rof e Ibu p
Cumulative drug permeated ( µ g/cm2 )
2000
ial
1200 ge l
200
Co mm erc
1400
og el
210
Ibuprofen emulsion gel Dexibuprofen emulsion gel Commercial hydrogel
0
1600
em u ls io n
220
1800
fen
Adhesiveness (g*sec)
230
*
*
2000
gel
Hardness (g)
240
up ro
*
3000
2500
2200
*
De x ib
250
Ib u p ro
Fig. 2 Hardness (a) and adhesiveness (b) of emulsion gels. *p \ 0.05 compared to commercial hydrogel. Each value represents the mean ± SD (n = 3)
obtained from the x-intercept at the steady-state permeated condition. The permeability coefficient (Kp) was calculated from the following steady-state equation: Kp = Mt/(Am * C0 * t), where Am, C0 and t were the exposure area of the skin sample (0.64 cm2), the initial drug concentration in the donor chamber (mM) and the time in hours, respectively (Gannu et al. 2010). Ibuprofen-loaded emulsion gel gave a significant higher permeability coefficient of about 1.7-fold compared to the commercial ibuprofen-loaded hydrogel, as the lag time of the former gel was nearly 0 h and that of the latter was about 2.2 h. Thus, the ibuprofenloaded emulsion gel improved the skin permeability. Ibuprofen-loaded emulsion gel could more readily solubilise the poorly water-soluble ibuprofen than the commercial hydrogel (Barot et al. 2012; Newa et al. 2008; Shah and D’mello 2007). Moreover, in general, a longer lag time was needed for the poorly water-soluble commercial hydrogel drug to permeate through the lipophilic skin (Sah and Bahl 2005). However, in the emulsion gel, the soluble drug in oil phase was rapidly permeated in the lipophilic skin (Murdan 2005). Furthermore, Tween 80 and isopropyl myristate used in the preparation of this emulsion gel might enhance the skin permeation (Song et al. 2009; Zhao et al. 2013), resulting in the lag time of nearly 0 h. On the other hand, the dexibuprofen-loaded emulsion gel showed about twofold skin permeability coefficient compared to ibuprofenloaded emulsion gel. This emulsion gel gave no lag time, like the ibuprofen-loaded emulsion gel. In particular, the
Mechanical properties, skin permeation 21
Table 1 Permeability parameters Lag time (h)
Permeability coefficient (9103 cm/h)
Commercial hydrogel
Linearity (r2)
20.5 ± 3.2
2.24 ± 0.34
0.972
Ibuprofen emulsion gel
35.2 ± 4.0*
0
0.993
Dexibuprofen emulsion gel
71.5 ± 4.9#
0
0.988
Each value represents the mean ± SD (n = 5) * p \ 0.05 compared to commercial hydrogel #
p \ 0.05 compared to commercial hydrogel and ibuprofen emulsion gel
Increase of pae volume (%)
*, #
+
**
15
**
12
Control Ibuprofen emulsion gel Dexibuprofen emulsion gel Commercial hydrogel
9
0.8
6
0.6
0 ** **
**
*
** ** ***, ##
0.2
** ***, +
Control ***, #, ++ Ibuprofen emulsion gel Dexibuprofen emulsion gel Commercial hydrogel
0.0 2
4
2
4
6
8
10
Time (h)
** *, ##
0.4
*. #
18
Nociceptive threshold (g)
Parameter
6
8
10
Time (h) Fig. 4 Anti-inflammatory efficacy. Anti-inflammatory efficacy, the increase of paw volume was calculated by the following equation: (Vt - V0)/V0 * 100 where V0 and Vt are volumes of the left hind paws at 0 and t h, respectively. *p \ 0.05, **p \ 0.01 and ***p \ 0.001 compared to control group. #p \ 0.05 and ##p \ 0.01 compared to commercial hydrogel group. ?p \ 0.05 and ??p \ 0.01 compared to ibuprofen emulsion gel group. Each value represents the mean ± SD (n = 6)
dexibuprofen-loaded emulsion gel enhanced skin permeability by about 3.5-fold without any lag time compared to the commercial hydrogel. The anti-inflammatory efficacy of dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel was assessed by the carrageenan-induced paw oedema test in rat’s hind paws compared to the commercial hydrogel (Bhandare et al. 2010; Kang et al. 2010; Lim et al. 2008). The increase in paw volume (%) was calculated using the following equation: (Vt - V0)/V0*100, where V0 and Vt are volumes of the left hind paws at 0 and t h, respectively (Kang et al. 2010; Patel et al. 2009). Until day 7, the paw volume was increased for control (no treatment) and then decreased after 10 days (Fig. 4), indicating self-healing. Three preparations
Fig. 5 Anti-nociceptive efficacy: Anti-nociceptive efficacy, nociceptive threshold was determined from the minimal weights (g) that caused each rat to withdraw its paw. *p \ 0.05 and **p \ 0.01 compared to control group. #p \ 0.05 compared to commercial hydrogel group. ?p \ 0.05 compared to ibuprofen emulsion gel. Each value represents the mean ± SD (n = 6)
increased the paw volume at 4 days, followed by a decreased volume at 7 days and a more decreased volume compared to the control group, indicating effective anti-inflammatory efficacy. At 7 days, the dexibuprofen emulsion gel gave the most decreased paw volume among all of the compounds. Furthermore, the dexibuprofen emulsion gel and the commercial hydrogel showed significantly more decreased paw volume than the ibuprofen emulsion gel at 10 days. Our results suggest that the anti-inflammatory efficacy, inhibiting effect of carrageenan-induced inflammation, was in the order of dexibuprofen emulsion gel [ commercial hydrogel [ ibuprofen emulsion gel. In particular, the dexibuprofen-loaded emulsion gel provided about twofold more antiinflammatory efficacy than control (0.642 ± 0.039 vs. 0.301 ± 0.015 % at 7 days; 0.365 ± 0.022 vs. 0.181 ± 0.009 % at 10 days). In addition, their anti-nociceptive efficacy was evaluated by the carrageenan-induced paw pressure test in rat’s hind paws. Nociceptive threshold (g), the force applied to the dorsal surface of the inflamed hind paw, was determined from the minimal weights (g) that caused each rat to withdraw its paw (Kang et al. 2010; Rutten et al. 2011; Makuch et al. 2013). Control (no treatment) gradually lowered the nociceptive threshold and paw withdrawal threshold to 10 days (Fig. 5), suggesting no anti-nociceptive efficacy. The commercial hydrogel gave constant thresholds of about 12 g, which was very similar to the
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control group up to 7 days. Therefore, this commercial hydrogel showed weak anti-nociceptive efficacy. The ibuprofen emulsion gel had similar thresholds until day 4 and increased thresholds compared to the control. However, for the test period, the dexibuprofen emulsion gel increased the thresholds compared to the control group and provided higher thresholds than the other preparations, leading to the most improved nociceptive threshold. Thus, the dexibuprofen-loaded emulsion gel gave excellent anti-inflammatory and anti-nociceptive efficacy due to its rapid skin permeation.
Conclusion In conclusion, the dexibuprofen-loaded emulsion gel with good mechanical properties, fast skin permeation, and excellent anti-inflammatory and anti-nociceptive efficacy would be an excellent candidate for topical delivery. Acknowledgments This work was supported by the research fund of Hanyang University (HY-2013-N).
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RESEARCH ARTICLE
Mechanical properties, skin permeation and in vivo evaluations of dexibuprofen-loaded emulsion gel for topical delivery Sung Giu Jin • Abid Mehmood Yousaf • Mi Woon Son Sun Woo Jang • Dong Wuk Kim • Jong Oh Kim • Chul Soon Yong • Jeong Hoon Kim • Han-Gon Choi
•
Received: 16 January 2014 / Accepted: 9 March 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract The aim of this research was to evaluate the gel properties, skin permeation and in vivo drug efficacy of a novel dexibuprofen-loaded emulsion gel for topical delivery. In this study, the dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel were prepared with isopropanol, Tween 80, propylene glycol, isopropyl myristate and carbopol. Their mechanical properties such as hardness and adhesiveness were assessed. Moreover, their skin permeation, anti-inflammatory and anti-nociceptive efficacy were evaluated using Franz diffusion cell with the hairless mouse skin, the carrageenan-induced paw oedema test and paw pressure test in rat’s hind paws compared with the commercial hydrogel, respectively. The dexibuprofen emulsion gel and ibuprofen emulsion gel provided significantly higher hardness and adhesiveness than the commercial hydrogel. The dexibuprofen emulsion gel enhanced skin permeability by about twofold and 3.5-fold without lag time compared to the ibuprofen emulsion gel and the commercial hydrogel, respectively, suggesting its faster skin permeation. Moreover, the anti-inflammatory efficacy and alleviation in carrageenan-induced inflammation was in the order of dexibuprofen emulsion gel [ commercial hydrogel [ ibuprofen emulsion gel. The dexibuprofen S. G. Jin M. W. Son S. W. Jang J. H. Kim (&) Pharmaceutical Product Research Laboratories, Dong-A Pharm. Co. Ltd., Yongin-Si, Kyunggi-Do 449-905, South Korea e-mail: [email protected] S. G. Jin A. M. Yousaf D. W. Kim H.-G. Choi (&) College of Pharmacy, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-791, South Korea e-mail: [email protected] J. O. Kim C. S. Yong College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyongsan 712-749, South Korea
emulsion gel furnished significantly higher nociceptive thresholds than the ibuprofen emulsion gel and the commercial hydrogel, leading to the most improved anti-nociceptive efficacy. Thus, this dexibuprofen-loaded emulsion gel with good mechanical property, rapid skin permeation and excellent anti-inflammatory and anti-nociceptive efficacy would be a strong candidate for the topical delivery of anti-inflammatory dexibuprofen. Keywords Dexibuprofen Emulsion gel Hardness Adhesiveness Skin permeation Anti-inflammatory efficacy Anti-nociceptive efficacy
Introduction Topical non-steroidal anti-inflammatory drugs (NSAIDs) are usually applied as a gel or a patch on the skin in order to reach the deep subcutaneous tissues, show direct efficacy and avoid adverse effects (Baek et al. 2013; Rhee et al. 2013). These drugs inhibit cyclooxygenases and activate peroxisome proliferators-activated receptors, leading to reduced inflammation (Cox et al. 1999). Ibuprofen, an equal mixture of the R(-)-enantiomer and S(?)-enantiomer, has been widely used in many countries because it presents minimum side effects involving gastric damage seen for many NSAIDs. However, R(-)-ibuprofen undergoes ‘‘metabolic inversion’’ to the extent of 57–69 % to yield S(?)-ibuprofen. Dexibuprofen, S(?)-isomer ibuprofen, is pharmacologically more effective than ibuprofen; since the former reduced the level of gastric damage and improved the analgesic effects in comparison to the latter, this suggests that the oral and gel forms have excellent antiinflammatory effect (Bonabello et al. 2003; Cox et al. 1999; Kaehler et al. 2003).
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Recently, the gel product has been given the brand name NurofenÒ (Reckitt Benckiser GmbH; Mannheim, Germany) (Sabale and Vora 2012; Khan et al. 2011). In this study, in order to develop a novel dexibuprofen emulsion gel with better anti-inflammatory efficacy than ibuprofen emulsion gel, this gel was prepared with isopropanol, Tween 80, propylene glycol, isopropyl myristate and carbopol. The emulsion gel form provides more solubility and skin-permeability of poorly water-soluble drugs than the hydrogel form (Barot et al. 2012; Murdan 2005). Therefore, the emulsion gel form was developed for dexibuprofen. Its mechanical properties such as hardness and adhesiveness were assessed. Moreover, its skin permeation, anti-inflammatory and anti-nociceptive efficacy were evaluated using Franz diffusion cell with the hairless mouse skin, the carrageenan-induced paw oedema test and paw pressure test in rat’s hind paws and compared with results of the commercial hydrogel.
and isopropyl myristate at the weight ratio of 5:10:10:5, respectively. Carbopol (2.5 g) was also dissolved in 60.3 g of distilled water. Two solutions and diisopropanolamine (2.2 g) was entirely mixed and emulsified using a homogeniser (T-50 basic Ultra Turrax; IKA-Werke GmbH, Staufen, Germany), leading to the formation of 100 g emulsion gels. Mechanical properties of emulsion gel
Materials and methods
The mechanical properties of emulsion gel such as hardness and adhesiveness were investigated using a Texture Analyser (TA-XT2Ò Texture Analyzer; Microsystems, Haslemere, Surrey, UK). In this investigation, the hemispherical analytical probe (diameter 1 cm) was twice compressed into each sample at a defined rate (1 mm/s) and a trigger force of 0.1 N to a depth of 15 mm. From the resultant force–time plot (Fig. 1), the hardness and adhesiveness values were obtained from maximum force and the area of the negative region of curve, respectively (Bansal et al. 2009).
Materials
Skin permeation
Dexibuprofen and ibuprofen were supplied from Dong-A Pharm. Co. (Suwon, South Korea) and were of USP grade. Isopropanol, polysorbate 80 (Tween 80), propylene glycol, isopropyl myristate and diisopropanolamine were bought from the DC Chemical Co. (Seoul, South Korea). Carbopol (934P) was supplied from BF Goodrich (Brecsville, OH, USA). The commercial product (NurofenÒ; in hydrogel type, 5 % w/w ibuprofen) was purchased from Reckitt Benckiser GmbH (Mannheim, Germany). All other chemicals were of reagent grade and used without further purification.
All of the skin permeation experiments were conducted using Franz diffusion cells (FCDV-15, Labfine; Anyang, South Korea). The capacities of the donor and the receiver chambers were 1 and 5.5 mL, respectively. The surface area available for diffusion was 0.64 cm2. The receiver chamber temperature was maintained at 36.5 ± °C using a flow loop consisting of a water bath. The receptor chambers of the Franz diffusion cells were filled with phosphate buffer solution (pH 7.4) (5.5 ml) with continuous stirring using a magnetic stirrer. The hairless mouse skin was thawed at room temperature for a minimum of 2 h and was placed between the donor and receiver chambers with the stratum corneum facing the donor chamber. Then, three preparations, such as dexibuprofen-loaded emulsion gel, ibuprofen-loaded emulsion gel and the commercial hydrogel at a drug dose of 25 mg, were placed inside the donor chambers of Franz diffusion cells. At predetermined time intervals, 0.4 ml samples were withdrawn from the receptor chamber using a syringe. Fresh phosphate buffer solution (0.4 ml) was replaced in the receptor chambers. The collected samples were stored at 4 °C until further analysis. The concentrations of dexibuprofen (or ibuprofen) in the receptor chambers (10 ll) were measured using a HPLC system (Shimadzu, Japan) consisting of Class VP computer software, LC 10AD VP pump and SPD 10A VP UVVIS detector. The column was an Inertsil ODS-3 C18 column (5 lm, 150 9 4.6 mm). The mobile phase, a mixture of phosphate buffer (pH 3.5) and acetonitrile (4:6 v/v), was filtered through 0.45 lm membrane filter and
Animal Male Sprague–Dawley rats (7–9 weeks old, weighing 200–250 g) and hairless mice (8–10 weeks old) were acquired from the Charles River Company Korea (Orient, Seoul, Korea). All animal care and experimentations were done in accordance with the Guiding Principles in the Use of Animals in Toxicology, as accepted in 1989, reviewed in 1999, and modified in 2008 by the Society of Toxicology (SOT 2008). The criteria for the use of animals in research were also permitted by the Institutional Animal Care and Use Committees of Dong-A Pharmaceutical Corporation. Preparation of emulsion gel Dexibuprofen (5 g) or ibuprofen (5 g) was dissolved in a 30 g mixture of isopropanol, Tween 80, propylene glycol
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Mechanical properties, skin permeation Fig. 1 Mechanical force of dexibuprofen-loaded emulsion gels. Hardness and adhesiveness was obtained from maximum force and the area of the negative region of the curve, respectively
eluted at a flow rate of 1 ml/min. Effluents were monitored at 220 nm (ibuprofen, 214 nm) (Baek et al. 2012; Balakrishnan et al. 2009). In vivo efficacy tests The anti-inflammatory and anti-nociceptive efficacy of dexibuprofen-loaded emulsion gel were assessed by the carrageenan-induced paw oedema test and paw pressure test in rat hind paws, respectively (Bhandare et al. 2010; Kang et al. 2010). The suspension of 1 % carrageenan in saline (100 ll) was injected into the plantar side of the left hind paw of the rat, followed by percutaneous administration of three preparations such as dexibuprofen-loaded emulsion gel, ibuprofen-loaded emulsion gel and commercial product at the drug dose of 25 mg/kg after 1 h. Carrageenan caused visible redness and pronounced swelling that was well-developed 1 h after the injection. No treatment was used as a control. Paw volume was measured at 0, 2, 4, 7 and 10 h after percutaneous administration using a plethysmometer (Ugo Basile; Comerio, Italy). Furthermore, at 0, 2, 4, 7 and 10 h, the left hind paw of each rat was placed onto an analgesia meter (Ugo Basile; Comerio, Italy). Then, the weights were increased until the rat withdrew its paw. The nociceptive threshold (g), the force applied to the dorsal surface of the inflamed hind paw, was determined from the minimal weight (g) that caused each rat to withdraw its paw (Kang et al. 2010; Makuch et al. 2013).
Results and discussion Dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel were prepared by mixing and homogenising an oil phase and a water phase. For poorly water-soluble drugs, the emulsion gel form provided more solubility and permeability than the hydrogel form (Barot et al. 2012; Murdan 2005). Accordingly, the dexibuprofen emulsion
gel form was developed. In this study, isopropanol, Tween 80 and diisopropanolamine were used as an oil, surfactant and co-surfactant, respectively (Balakrishnan et al. 2009; Khan et al. 2011; Yerramsetty et al. 2010). In addition, carbopol was used as a thickening agent (Sheshala et al. 2013). In a preliminary study, carbopol provided stronger mechanical force to the emulsion gels compared to other hydrophilic polymers such as HPC, PVP and HPMC (data not shown). Then, their mechanical forces, such as hardness and adhesiveness, were investigated and compared with the commercial product. The commercial product (NurofenÒ) was a 5 % ibuprofen-loaded hydrogel prepared with carbopol as a base (Rhee et al. 2008; Serikov 2007). Hardness, which means the force required to attain a given deformation, was found from maximum force (Bansal et al. 2009). Furthermore, adhesiveness, which is the work required to overcome the attractive forces between the surface of the sample and the surface of the probe, was calculated from the area of the negative region of the curve (Fig. 1). Dexibuprofen-loaded emulsion gel and ibuprofenloaded emulsion gel gave significantly higher hardness (Fig. 2a) and adhesiveness (Fig. 2b) than the commercial hydrogel (243 ? 4 vs. 243 ? 5 vs. 227 ? 4 g; 1,999 ? 35 vs. 1,959 ? 36 vs. 1,810 ? 22 g s). However, there were no significant differences in the values between dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel. Thus, the emulsion gels prepared in this study had the excellent mechanical force compared to the commercial hydrogel due to the use of carbopol, a very highly viscous polymer (Sheshala et al. 2013). Moreover, the emulsion was more viscous than the solution. Therefore, the emulsion gel provided more viscosity than the general hydrogel with a single water phase (Murdan 2005). Next, the skin permeation study was performed using Franz diffusion cell with hairless mouse skin compared to the commercial hydrogel. From Fig. 3, the cumulative amounts of drug permeated (Mt, lg/cm2) were calculated as the total amount of drug permeated through the skin during a time period of 24 h (Gannu et al. 2010). Dexibuprofen-loaded emulsion gel and ibuprofen-loaded
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(A)
(B)
1000
500
0 6
12
18
24
Time (h) Fig. 3 Permeation profiles of drug in the emulsion gels. Each value represents the mean ± SD (n = 5)
emulsion gel showed significantly cumulative amounts of drug permeation compared to the commercial ibuprofenloaded hydrogel. The cumulative amounts of drug permeated from dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel were not significantly different. Table 1 shows their permeability parameters, such as permeability coefficient and lag time. The lag times (Lt) were
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el hyd rog
ge l em u ls io n fe n
ial
hy dr
gel fen
upr o De x ib
1500
Co mm erc
em u ls io n
mu lsio n ne rof e Ibu p
Cumulative drug permeated ( µ g/cm2 )
2000
ial
1200 ge l
200
Co mm erc
1400
og el
210
Ibuprofen emulsion gel Dexibuprofen emulsion gel Commercial hydrogel
0
1600
em u ls io n
220
1800
fen
Adhesiveness (g*sec)
230
*
*
2000
gel
Hardness (g)
240
up ro
*
3000
2500
2200
*
De x ib
250
Ib u p ro
Fig. 2 Hardness (a) and adhesiveness (b) of emulsion gels. *p \ 0.05 compared to commercial hydrogel. Each value represents the mean ± SD (n = 3)
obtained from the x-intercept at the steady-state permeated condition. The permeability coefficient (Kp) was calculated from the following steady-state equation: Kp = Mt/(Am * C0 * t), where Am, C0 and t were the exposure area of the skin sample (0.64 cm2), the initial drug concentration in the donor chamber (mM) and the time in hours, respectively (Gannu et al. 2010). Ibuprofen-loaded emulsion gel gave a significant higher permeability coefficient of about 1.7-fold compared to the commercial ibuprofen-loaded hydrogel, as the lag time of the former gel was nearly 0 h and that of the latter was about 2.2 h. Thus, the ibuprofenloaded emulsion gel improved the skin permeability. Ibuprofen-loaded emulsion gel could more readily solubilise the poorly water-soluble ibuprofen than the commercial hydrogel (Barot et al. 2012; Newa et al. 2008; Shah and D’mello 2007). Moreover, in general, a longer lag time was needed for the poorly water-soluble commercial hydrogel drug to permeate through the lipophilic skin (Sah and Bahl 2005). However, in the emulsion gel, the soluble drug in oil phase was rapidly permeated in the lipophilic skin (Murdan 2005). Furthermore, Tween 80 and isopropyl myristate used in the preparation of this emulsion gel might enhance the skin permeation (Song et al. 2009; Zhao et al. 2013), resulting in the lag time of nearly 0 h. On the other hand, the dexibuprofen-loaded emulsion gel showed about twofold skin permeability coefficient compared to ibuprofenloaded emulsion gel. This emulsion gel gave no lag time, like the ibuprofen-loaded emulsion gel. In particular, the
Mechanical properties, skin permeation 21
Table 1 Permeability parameters Lag time (h)
Permeability coefficient (9103 cm/h)
Commercial hydrogel
Linearity (r2)
20.5 ± 3.2
2.24 ± 0.34
0.972
Ibuprofen emulsion gel
35.2 ± 4.0*
0
0.993
Dexibuprofen emulsion gel
71.5 ± 4.9#
0
0.988
Each value represents the mean ± SD (n = 5) * p \ 0.05 compared to commercial hydrogel #
p \ 0.05 compared to commercial hydrogel and ibuprofen emulsion gel
Increase of pae volume (%)
*, #
+
**
15
**
12
Control Ibuprofen emulsion gel Dexibuprofen emulsion gel Commercial hydrogel
9
0.8
6
0.6
0 ** **
**
*
** ** ***, ##
0.2
** ***, +
Control ***, #, ++ Ibuprofen emulsion gel Dexibuprofen emulsion gel Commercial hydrogel
0.0 2
4
2
4
6
8
10
Time (h)
** *, ##
0.4
*. #
18
Nociceptive threshold (g)
Parameter
6
8
10
Time (h) Fig. 4 Anti-inflammatory efficacy. Anti-inflammatory efficacy, the increase of paw volume was calculated by the following equation: (Vt - V0)/V0 * 100 where V0 and Vt are volumes of the left hind paws at 0 and t h, respectively. *p \ 0.05, **p \ 0.01 and ***p \ 0.001 compared to control group. #p \ 0.05 and ##p \ 0.01 compared to commercial hydrogel group. ?p \ 0.05 and ??p \ 0.01 compared to ibuprofen emulsion gel group. Each value represents the mean ± SD (n = 6)
dexibuprofen-loaded emulsion gel enhanced skin permeability by about 3.5-fold without any lag time compared to the commercial hydrogel. The anti-inflammatory efficacy of dexibuprofen-loaded emulsion gel and ibuprofen-loaded emulsion gel was assessed by the carrageenan-induced paw oedema test in rat’s hind paws compared to the commercial hydrogel (Bhandare et al. 2010; Kang et al. 2010; Lim et al. 2008). The increase in paw volume (%) was calculated using the following equation: (Vt - V0)/V0*100, where V0 and Vt are volumes of the left hind paws at 0 and t h, respectively (Kang et al. 2010; Patel et al. 2009). Until day 7, the paw volume was increased for control (no treatment) and then decreased after 10 days (Fig. 4), indicating self-healing. Three preparations
Fig. 5 Anti-nociceptive efficacy: Anti-nociceptive efficacy, nociceptive threshold was determined from the minimal weights (g) that caused each rat to withdraw its paw. *p \ 0.05 and **p \ 0.01 compared to control group. #p \ 0.05 compared to commercial hydrogel group. ?p \ 0.05 compared to ibuprofen emulsion gel. Each value represents the mean ± SD (n = 6)
increased the paw volume at 4 days, followed by a decreased volume at 7 days and a more decreased volume compared to the control group, indicating effective anti-inflammatory efficacy. At 7 days, the dexibuprofen emulsion gel gave the most decreased paw volume among all of the compounds. Furthermore, the dexibuprofen emulsion gel and the commercial hydrogel showed significantly more decreased paw volume than the ibuprofen emulsion gel at 10 days. Our results suggest that the anti-inflammatory efficacy, inhibiting effect of carrageenan-induced inflammation, was in the order of dexibuprofen emulsion gel [ commercial hydrogel [ ibuprofen emulsion gel. In particular, the dexibuprofen-loaded emulsion gel provided about twofold more antiinflammatory efficacy than control (0.642 ± 0.039 vs. 0.301 ± 0.015 % at 7 days; 0.365 ± 0.022 vs. 0.181 ± 0.009 % at 10 days). In addition, their anti-nociceptive efficacy was evaluated by the carrageenan-induced paw pressure test in rat’s hind paws. Nociceptive threshold (g), the force applied to the dorsal surface of the inflamed hind paw, was determined from the minimal weights (g) that caused each rat to withdraw its paw (Kang et al. 2010; Rutten et al. 2011; Makuch et al. 2013). Control (no treatment) gradually lowered the nociceptive threshold and paw withdrawal threshold to 10 days (Fig. 5), suggesting no anti-nociceptive efficacy. The commercial hydrogel gave constant thresholds of about 12 g, which was very similar to the
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control group up to 7 days. Therefore, this commercial hydrogel showed weak anti-nociceptive efficacy. The ibuprofen emulsion gel had similar thresholds until day 4 and increased thresholds compared to the control. However, for the test period, the dexibuprofen emulsion gel increased the thresholds compared to the control group and provided higher thresholds than the other preparations, leading to the most improved nociceptive threshold. Thus, the dexibuprofen-loaded emulsion gel gave excellent anti-inflammatory and anti-nociceptive efficacy due to its rapid skin permeation.
Conclusion In conclusion, the dexibuprofen-loaded emulsion gel with good mechanical properties, fast skin permeation, and excellent anti-inflammatory and anti-nociceptive efficacy would be an excellent candidate for topical delivery. Acknowledgments This work was supported by the research fund of Hanyang University (HY-2013-N).
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