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Preparation, charactarization and anti-inflammatory activity of celecoxib chitosan gel formulations
J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
Preparation, charactarization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry*1, 2, G. Fetih2 Department of Pharmaceutics, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia 2 Department of Pharmaceutics, Faculty of Pharmacy, Assiut University 71526, Assiut, Egypt *Correspondence: [email protected]
1
This study was designed to evaluate the suitability of chitosan polymer as a vehicle for topical delivery system. Celecoxib, which is a nonsteroidal anti-inflammatory drug, was incorporated into the gel vehicles in a concentration of 0.5 % w/v. Gels were prepared using three different concentrations and different molecular weights of chitosan. Viscosity, drug release from gels, permeation of drug through rat skin and anti-inflammatory activity of the drug were studied. In vitro release characteristics of the drug from different gels were carried out using dialysis membrane in phosphate buffer using a pH of 6.8. The results showed that, the gel form containing 1.0 % w/v medium molecular weight chitosan has superior drug release than other forms, whilst the gel form containing 2.0 % w/v high molecular weight chitosan shows the lowest amount of drug release. The release data were treated with various kinetic principles to assess the relevant parameters. The results revealed an inverse correlation between the percent drug release and the polymer concentration used. The results also showed that the release of drug from the prepared gels obeyed the Higuchi’s diffusion model. The permeation of drug through rat skin was carried out. The flux of drug is independent on the viscosity of the formulae. The anti-inflammatory activity of the drug in different gel formulations was studied using carrageenan-induced rat paw edema method. The results obtained show that there is excellent anti-inflammatory activity of the gel forms on rat paw edema. Key words: Celecoxib – Chitosan gel formulations – In vitro release – Kinetic study – Permeation through skin – Anti-inflammatory activity.
Non-steroidal, anti-inflammatory drugs (NSAIDs) are widely used to provide effective therapy in patients with chronic rheumatic disorders. However, their oral administration has been associated with a number of gastrointestinal disorders which can reduce patient compliance. celecoxib, a selective COX-2 inhibitor, has been approved for the treatment of rheumatoid arthritis, osteoarthritis, acute pain, familial polyposis and primary dysmenorrhea [1-3]. In this study, celecoxib was used as model of the COX-2 drugs. Moreover, topical formulations of COX-2 treated some skin diseases [4]. Researchers have been trying to overcome gastrointestinal side effects by topical delivery of NSAIDs. Skin has been shown to be a suitable delivery route for drugs formulated topically during the past few years. Ketoprofen, naproxen, tenoxicam, meloxicam and celecoxib can be counted among these drugs [5-8]. Chitosan, a polysaccharide derived from naturally abundant chitin, is currently receiving a great deal of attention for medical and pharmaceutical applications. The main reasons for this increasing interest are undoubtedly due to its appealing intrinsic properties. Indeed, chitosan is known for its biocompatibility, allowing its use in various medical applications such as topical ocular application [9], implantation [10] or injection [11]. Moreover, chitosan is metabolized by certain human enzymes, e.g. Lysozyme, and can be considered as biodegradable [12, 13]. In addition, it has been reported that chitosan acts as a penetration enhancer [14]. Chitosan also promotes wound-healing [15] and has bacteriostatic effects [16]. Finally, chitosan is abundant in nature, and its production is of low cost and is ecologically interesting [17]. In Pharmaceutical applications, chitosan is used as a component in hydrogel. The main parameters influencing the characteristics of chitosan are its molecular weight (MW) and its degree of deacetylation [15]. The aim of the present study is the preparation of celecoxib in different chitosan gel formulations and studies of the effect of the concentration of chitosan and its molecular weight on the in vitro release of the drug and its permeation through rat skin. Another aim is to study the performance of the gel formulations on the anti-inflammatory activity of the paw edema in rats.
I. MATERIALS AND METHODS 1. Materials
Celecoxib was kindly donated by Searle, Augusta GA, United States. Chitosan with differing molecular weights came from Fluka Chemie AG (Buchs, Switzerland) and semi-permeable membrane, cellulose in nature with cutoff 1000 Dalton (Cellulose tubing, Sigma Diagnostics, St. Louis, MO, United States). All other chemical were of analytical grade.
2. Preparation of chitosan gels
Chitosan gels were prepared at 1-3 % w/v of the medium molecular weight, 2 % w/v of low, medium and high molecular weight in lactic acid solution (1.0 % v/v). 0.5 % w/v of drug was dissolved in the least amount of propylene glycol and added to the chitosan gel. Methyl paraben sodium salt (0.1 %, w/v) as preservative was added. The contents were stirred and the resulting gel solutions were sonicated to remove the air bubbles. Each of the test samples was transferred into 4-oz tubes till further use.
3. Determination of gel viscosity
Rheological experiments were performed to examine the viscous and elastic properties of the different formulations. Viscosity measurements of gels were performed on a Brookfield Model DV-II+ digital viscometer (Brookfield Engineering Laboratories, INC, Stoughton, United States) at room temperature.
4. In vitro release of celecoxib from gel formulations
In vitro release of the drug from different gel formulations were evaluated using semipermeable membrane as reported by El Maghraby et al. [18]. This employed the FDC-6 Transdermal Diffusion Cell Drive Console (Logan Instrument Corp., NJ, United States). The system is fitted with VTC-200 heater circulator with jacketed vertical glass Franz diffusion cells. The semipermeable cellulose membrane with cutoff 1000 Dalton (Cellulose tubing, Sigma diagnostics, St. Louis, MO, United States) was mounted between the donor and acceptor 201
J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
compartments of the diffusion cell. The receptor compartment was 12 mL and the cells have a diffusional area of 1.7 cm-1. The receptor cell was filled with phosphate buffer (pH 6.8). The system was adjusted to 37 ºC ± 0.5. 0.5 g of the tested formulations was loaded into the donor compartment and occluded using a parafilm. Aliquots (2.0 mL) were withdrawn at specific time intervals and replaced with fresh media. The samples were measured spectrophotometrically (UV-spectrophotometer, Schmidzu-50-02, Kyoto, Japan) at a maximum wave length of 252 nm [19] against similarly treated blanks. The percent cumulative amount of drug released was calculated. All experiments were carried out in triplicate and the average values were calculated.
8. Data analysis
The cumulative amounts of drug permeated with time produced the permeation profiles. These profiles were used to calculate the flux of the drug, which was obtained from the slope of the regression line fitted to the linear portion of the curve. Lag time was calculated from the extrapolation of this line x-axis.
9. Anti-inflammatory activity of the prepared gels
An acute inflammatory activity model, carrageenan-induced rat paw edema method was applied in this study [22, 23]. Measurements of the in vivo anti-inflammatory activity of the formulae conformed to guide lines of the Institutional Animal Ethical Committee of King Saud University. Thirty-six rats weighing about 200 g were divided into six groups of six rats. The animals of group 1 received placebo gel (chitosan gel without drug) and group 2, 3, 4, 5 and 6 received F1 (1.0 % medium molecular weight), F2 (2.0 % medium molecular weight), F3 ( 3.0 % medium molecular weight), F4 ( 2.0 % low molecular weight) and F5 (2.0 % high molecular weight) gel formulations, respectively. Inflammation was produced in the rats using 0.1 mL of 1.0 % w/v carrageenan solution in saline. This was injected subcutaneously into left hind paw. To evaluate the topical anti-inflammatory activity of the gel formulations, paw edema was examined. Thirty minutes later, 0.5 g of each gel was applied topically to the edematous paw. The increase in paw thickness was measured before carrageenan injection (time 0) and 2, 3, 4, 6 and 8 h after carrageenan administration using a dial micrometer. The percentage swelling of the paw and the percent inhibition of edema were calculated. The data were reported as mean ± SD (n = 6).
5. Kinetic treatment of the release data
The in vitro release data of the drug from the investigated gel formulations were studied by curve fitting method to different kinetic models of zero-order, first-order and Higuchi models. - Zero-order release: Mt/Mω = kt
Eq. 1
Ln ( 1- Mt/Mω) = - kt
Eq. 2
(Mt/Mω)2 = kt
Eq. 3
- First- order release:
- Higuchi model :
- The Korsmeyer-Peppas equation : Mt/Mω = ktn
10. Statistical analysis for the obtained results
Statistical analysis for the obtained results was carried out by the student t-test at a 0.05 level of significance.
Eq. 4
This was used to study drug release mechanism by analyzing n as the diffusion exponent. According to this equation, if n ≤ 0.45 a Fickian mechanism, if 0.5 ≤ n ≤ 0.8 a non-Fickian and if 0.8 ≤ n ≤ 1 a zero-order mechanism is governing the drug release mechanism from the gel [20].
II. RESULTS AND DISCUSSION
Chitosan as a non-toxic, biocompatible and biodegradable polymer has been widely used for pharmaceutical and medical application. In this work, Chitosan was used as gel forming agent to study the effect of chitosan concentration and its molecular weight on the release of the drug from the representative formulations. In the preliminary study, the lower molecular weight chitosan required a concentration above 1.7 % (w/v) to produce gel, so we used different concentration of the medium molecular weight polymer. Visual inspection of freshly prepared formulae revealed smooth homogenous topical preparations with acceptable spreadability.
6. Preparation of skin samples
A skin permeation study of celecoxib gel formulations was carried out using full thickness rat skin [21]. To obtain skin, male Wister rats weighing 200-250 g were used in this study. All experimental procedures were in accordance with the guidelines of the Institutional Animal Ethical Committee of King Saud University. Rates were anesthetized using sodium pentobarbitone (60 mg/kg) intraperitoneally. The abdominal skin was shaved using an animal hair clipper, in the direction of the tail to head. The skin was excised from the abdominal region of the sacrificed rats. The dermis part of the skin was wiped three times with a wet cotton swab soaked in isopropanol to remove any adhering fat material. Then, the skin was kept in normal saline solution for two hours. The cleaned skin was washed with distilled water, wrapped in aluminum foil, and stored in a deep freezer at - 20 °C until further use.
1. Viscosity study
It was seen from the flow curves (Figures 1 and 2) that celecoxib containing chitosan gels exhibit psudoplastic flow and the viscosity increases significantly with increasing either the chitosan concentration (Figure 1) or its molecular weight (Figure 2). The viscosity of 3.0 % (w/v) chitosan gel was found to be higher than that of 2.0 % (w/v) and 1.0 % (w/v). Also the viscosity of 2.0 % (w/v) chitosan gel prepared from a high molecular weight was found to be higher than that of medium and low molecular weight (Table I).
7. Skin permeation studies
As mentioned in the in vitro release experiments, skin permeation studies were carried out using an FDC-6 Transdermal Diffusion Cell Drive Console (Logan Instrument Corp., NJ, United States). The skin was mounted with the stratum corneal side uppermost on the vertical glass diffusion cells. The mounted skin was left to equilibrate overnight. 0.5 g of the tested formulations was loaded on the skin surface into the donor compartment and occluded with parafilm. Samples were withdrawn periodically and replaced with fresh medium. These samples were analyzed for drug concentration. All experiments were carried out in triplicate and the average values were calculated.
Table I - Relationship between the viscosities (cP) of the prepared chitosan gel formulations and the percent of celecoxib released after 4 h.
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Formulation code
Content
Viscosity (cP)
% celocoxib released after 4h
F1 F2 F3 F4 F5
1.0 % MMW 2.0 % MMW 3.0 % MMW 2.0 % LMW 2.0 % HMW
10000 ± 150 13700 ± 220 15250 ± 320 11500 ± 200 17150 ± 250
75 ± 2.2 66 ± 1.8 56 ± 1.5 71 ± 1.5 52 ± 2.2
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
100
60
90 80
% drug released
Shearing rate (s -1)
50 40 30 20 1.0 % (W/V)
10 0 0
500
1000
1500
70 60 50 40 30
2.0 % ( W/V)
20
3.0 % (W/V)
1.0 % (W/V) 2.0 % (W/V)
10
3.0 % (W/V)
2000
2500
0
3000
Shearing stress (dyne/cm2)
100
150
200
250
300
Figure 3 - Release profiles of celecoxib from chitosan gels prepared with different concentration of medium molecular weight chitosan. 100
60
90
% of drug released
50 -1
50
time (min)
Figure 1 - Flow curves of chitosan gels prepared with different concentration of medium molecular weight chitosan.
Shearing rate (s )
0
40 30 20 Low MW 10
Medium MW High MW 500
1000
1500
2000
2500
70 60 50 40 30
Low MW
20
Medium MW High MW
10
0 0
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0
3000
2
0
50
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time (min)
Shearing strees (dyne/cm ) Figure 2 - Flow curves of chitosan gels prepared with 2.0 % (w/v) of different molecular weight chitosan.
Figure 4 - Release profiles of celecoxib from chitosan gels prepared with 2.0 % (w/v) of different molecular weight chitosan.
2. In vitro drug release study
be related to the drug trapped between the tortuous ways of the gels which take longer to be released [20]. Viscosity of the gel formulation may be a very important factor that affects the release of drug since it may reduce diffusion rate of drug from the vehicles. Hereby, a reverse relationship was observed between the viscosity of chitosan gel and the percent of celecoxib released (Table I). The gel formulation containing 1.0 % (w/v) chitosan with a medium molecular weight has the lowest viscosity (10000 ± 150 cP) and showed the highest percent of drug released (75 ± 2.2). While 2.0 % (w/v) chitosan with a high molecular weight has the highest viscosity (17150 ± 250 cP) showed the lowest percent of drug release (52 ± 2.2).This result was in agreement with El-Maghraby [18], who is reported that the release rate of hydrocortisone is dependent on the viscosity of the microemulsion formulations. A similar finding was obtained by Hirano et al. [24] who concluded that, the higher the viscosity of the gel, the slower the drug released. Ismail et al. [25] observed that the release rate of carbachol chloride from carbopol 934 gel was greater than that from carbopol 940 gel. These results were attributed to the fact that cabopol 934 gel base exhibited a lower viscosity value than that of carbopol 940 gel base and so, it released the drug rapidly [26]. This is in accordance with the data obtained by Eros et al. [27] who studied the effect of viscosity on the drug release from different topical formulations. They found a reciprocal correlation between viscosity and the amount of the drug released. Release kinetic models are shown in Table II. As this table indicates
Drug release study from gel formulations is an important step during the development stages of topical formulation. To study the simultaneous effect of the molecular weight and the concentration of chitosan on the release profiles of drug, the percentage release of the drug from various chitosan gel formulations through a four hour period was calculated (Figure 3). Increasing the chitosan concentration of the medium molecular weight from 1.0 % (w/v) to 3.0 % (w/v) resulted in decreasing the percent of drug released (p < 0.05). With 1.0 % (w/v) gel formulation, 75 ± 2.2 % of celecoxib was released. For 2.0 % (w/v) chitosan gel, the percent release of drug was 66 ± 1.8 %. Increasing the gel forming agent to 3.0 % (w/v), the percentage of the drug was 56 ± 1.5 %. The effect of chitosan molecular weight on the release of drug from gel formulations was studied (Figure 4). The concentration of gel forming agent used was 2.0 %. There is an inversely proportional relationship between the molecular weight of chitosan used and the percent release of drug from prepared gels (p < 0.05). For the high molecular weight grade, the drug release was 52 ± 2.2 %, whereas the drug release was 66 ± 1.8 % and 71 ± 1.5 % for medium and low molecular weight grades. The results of the release test indicate that increasing the chitosan concentration or chitosan molecular weight reduces the release of drug as the penetration rate of water decreases in higher viscosities of the gels. All the drug release curves seem to follow biphasic pattern, with a faster rate of drug release over the first 90 min followed by a slower release for the remaining time that may 203
J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
the correlation coefficient of the release data fitted to Higuchi model which is higher than other models and all chitosan gel formulations, the diffusion exponent of Peppas equation is less than 0.5 which indicates a Fickian mechanism is dominant and controls the drug release from chitosan gels.
indicates that there was an inversely proportional relationship between the viscosity of the gel formulation and the flux of the drug. Also, the flux of celecoxib of F3 is higher than F4 and F5. Correlation between the skin permeation and the release results showed that the release rate was greater than the permeation flux of drug in all gel formulations. Hillton et al. [29] has reported that there are many factors that may influence the extent of percutaneous absorption of a drug. Partitioning of the chemical between the vehicle and the stratum corneum results in a concentration gradient developing across the skin, influenced by chemical-vehicle-skin interaction.
3. Drug permeation through rat skin
4. Anti-inflammatory activity studies using paw edema
Topical anti-inflammatory activity of semisolid preparations has been reported when applied 1 and 2 h before carrageenan treatment as mentioned by Hiramatsu et al. [30]. Clinically, it seems more reasonable to apply the anti-inflammatory topical preparations after the inflammation stimulus [23]. Table IV illustrates the anti-inflammatory activity of different gel formulations on the hind paw of the rats. It is shown that all the gel formulations have a significant effect (p < 0.05) as anti-inflammatory vehicle, but to a variable extent (inhibition
600
-1
Cumulative amount permeated (ug cm )
-2
Cumulative amount permeated (ug cm )
In vivo measurement of drug administered transdermally is a complex process. Due to the lack of availability of human skin, the in vitro drug permeation through skin was introduced as an alternative way to measure drug permeation. The Wister rat skin model has been used extensively, in view of the fact that its stratum corneum thickness, as well as water permeability, is the same as that of human skin [28]. In this study, drug permeation prior to steady state was similar for all gel formulations during the first hour, as it can be seen in Figures 5 and 6. After that the permeation is changed according to the content of gel formulations. From Table III, it was observed that the lag time for all formulae was between 0.5 and 0.75 h. The flux of celecoxib during the steady state of formula F3 was significantly higher than that of F2 and F1 that according to the viscosity of each formula. This
500 400 300 200 1.0 % (W/V)
100
2.0 % (W/V) 3.0 % (W/V)
0 0
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4
6
8
10
600
500
400
300
200 Low MW
100
Medium MW High MW
0
0
12
2
4
6
8
10
12
time (hr)
time (hr)
Figure 5 - Permeation profiles of celecoxib obtained after application of chitosan gels prepared with different concentration of medium molecular weight chitosan.
Figure 6 - Permeation profiles of celecoxib obtained after application of chitosan gels prepared with 2.0 % (w/v) of different molecular weight chitosan.
Table II - Kinetic models of celecoxib release from different chitosan gels. Table III - The permeation parameters of celecoxib obtained from different gel formulations.
Gel form
Zero-order (r)
First-order (r)
Higuchi model (r)
n
F1 F2 F3 F4 F5
0.856±0.025 0.900±0.003 0.909±0.010 0.841±0.015 0.925±0.010
0.929±0.015 0.960±0.010 0.950±0.023 0.920±0.025 0.953±0.021
0.980±0.011 0.987±0.002 0.991±0.001 0.984±0.002 0.992±0.004
0.44±0.02 0.45±0.03 0.48±0.01 0.44±0.02 0.45±0.03
Formulation code
Flux (µg cm-2 h-1)
Lag time (h)
F1 F2 F3 F4 F5
35.75 ± 0.35 48.35 ± 2.15 69.38 ± 3.25 34.30 ± 1.12 59.10 ± 2.50
0.75 ± 0.05 0.55 ± 0.03 0.50 ± 0.02 0.71 ± 0.13 0.61 ± 0.09
r: correlation coefficient. n: release exponent of Korsmeyer-Peppas equation. Table IV - Anti-inflammatory activity of celecoxib in different gel formulations. Rat group No.
Gel formulation
1 2 3 4 5 6
control F1 F2 F3 F4 F5
% swelling of induced edema 2h
3h
4h
6h
8h
95 ± 0.5 (5.00) 51.0±0.5 (48.8) 65.1±0.4 (35.9) 61.2±0.8 (39.0) 56.1±0.4 (44.9) 73.2±0.3 (27.8)
94.0 ± 0.5 (6.0) 40.1±0.6 (59.4) 60.2±2.0 (39.9) 54.3±0.5 (45.1) 51.5±2.0 (47.9) 65.3±0.2 (34.5)
93.0 ± 1.0 (7.2) 33.5±0.5 (65.6) 50.2±1.5 (50.2) 51.2±0.7 (49.5) 50.5±1.2 (50.5) 58.4±1.1 (42.3)
91.0 ± 0.6 (9.2) 30.3±0.4 (69.5) 45.3±0.9 (55.5) 45.1±0.5 (54.4) 40.3±1.2 (58.9) 52.6±1.3 (48.5)
90 ± 0.5 (10.0) 21.5±0.3 (79.5) 40.5±2.5 (59.5) 41.4±1.5 (58.6) 32.5±1.1 (67.5) 47.8±0.9 (53.3)
Mean ± SD. The value between parentheses indicates the % inhibition of edema. 204
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
percentage about 27.8-79.5) over time course studied (1-8 h) as compared with control. After the first 2 h of the observation, F1 chitosan gel shows 48.8 % inhibition while F5 chitosan gel was 27.8 %. After 6 h, the inhibitory effect was 79.5 and 53.3 for the same chitosan gel forms, respectively. The rank order of edema inhibition using this method was in the following order F1 > F4 > F2 > F3 > F5. This order is similar to that appearing in the percent of drug release. This evident a strong correlation between the percent of drug release from the various chitosan gel formulations and the anti-inflammatory activity. Correlating the in vitro release results (Table I), permeation results (Table III) and anti-inflammatory activity (Table IV). The formulation were ranked in the term of release rate and anti-inflammatory activity as F1 > F4 > F2 > F3 > F5. In terms of skin permeation they were ranked as F3 > F5 > F2 > F1 > F4. This evident taking into consideration that the release rates of drug were greater than the transdermal flux in all cases. In addition, there were no significant differences between the lag time values obtained after application of different formulations. The lag time is a permeation parameter depending mainly on the diffusivity of the drug through the skin. In this study, by increasing the permeation of drug through the skin, the localization of drug in epidermal layer decreases so the anti-inflammatory activity decreases and vice versa.
10. 11.
12. 13. 14.
15. 16.
*
17.
The results of in vitro release, permeation and anti-inflammatory studies concluded that, a chitosan is a good carrier for celecoxib as gel formulation. The polymer concentration and the molecular weight of chitosan are very effective factors on the release of drug from gel formulations. Celecoxib permeation through rat skin is independent of the viscosity of the gel. All gel formulations have significant antiinflammatory activity.
18. 19. 20. 21.
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J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
AcknowledgmentS
Manuscript
The authors would like to thank Dr. Mohamed A. Ibrahim for his advices during rewriting of the manuscript. Also, we would like to appreciate Dr. M. Kazie for revision the manuscript.
Received 6 December 2010, accepted for publication 14 March 2011.
206
Preparation, charactarization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry*1, 2, G. Fetih2 Department of Pharmaceutics, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia 2 Department of Pharmaceutics, Faculty of Pharmacy, Assiut University 71526, Assiut, Egypt *Correspondence: [email protected]
1
This study was designed to evaluate the suitability of chitosan polymer as a vehicle for topical delivery system. Celecoxib, which is a nonsteroidal anti-inflammatory drug, was incorporated into the gel vehicles in a concentration of 0.5 % w/v. Gels were prepared using three different concentrations and different molecular weights of chitosan. Viscosity, drug release from gels, permeation of drug through rat skin and anti-inflammatory activity of the drug were studied. In vitro release characteristics of the drug from different gels were carried out using dialysis membrane in phosphate buffer using a pH of 6.8. The results showed that, the gel form containing 1.0 % w/v medium molecular weight chitosan has superior drug release than other forms, whilst the gel form containing 2.0 % w/v high molecular weight chitosan shows the lowest amount of drug release. The release data were treated with various kinetic principles to assess the relevant parameters. The results revealed an inverse correlation between the percent drug release and the polymer concentration used. The results also showed that the release of drug from the prepared gels obeyed the Higuchi’s diffusion model. The permeation of drug through rat skin was carried out. The flux of drug is independent on the viscosity of the formulae. The anti-inflammatory activity of the drug in different gel formulations was studied using carrageenan-induced rat paw edema method. The results obtained show that there is excellent anti-inflammatory activity of the gel forms on rat paw edema. Key words: Celecoxib – Chitosan gel formulations – In vitro release – Kinetic study – Permeation through skin – Anti-inflammatory activity.
Non-steroidal, anti-inflammatory drugs (NSAIDs) are widely used to provide effective therapy in patients with chronic rheumatic disorders. However, their oral administration has been associated with a number of gastrointestinal disorders which can reduce patient compliance. celecoxib, a selective COX-2 inhibitor, has been approved for the treatment of rheumatoid arthritis, osteoarthritis, acute pain, familial polyposis and primary dysmenorrhea [1-3]. In this study, celecoxib was used as model of the COX-2 drugs. Moreover, topical formulations of COX-2 treated some skin diseases [4]. Researchers have been trying to overcome gastrointestinal side effects by topical delivery of NSAIDs. Skin has been shown to be a suitable delivery route for drugs formulated topically during the past few years. Ketoprofen, naproxen, tenoxicam, meloxicam and celecoxib can be counted among these drugs [5-8]. Chitosan, a polysaccharide derived from naturally abundant chitin, is currently receiving a great deal of attention for medical and pharmaceutical applications. The main reasons for this increasing interest are undoubtedly due to its appealing intrinsic properties. Indeed, chitosan is known for its biocompatibility, allowing its use in various medical applications such as topical ocular application [9], implantation [10] or injection [11]. Moreover, chitosan is metabolized by certain human enzymes, e.g. Lysozyme, and can be considered as biodegradable [12, 13]. In addition, it has been reported that chitosan acts as a penetration enhancer [14]. Chitosan also promotes wound-healing [15] and has bacteriostatic effects [16]. Finally, chitosan is abundant in nature, and its production is of low cost and is ecologically interesting [17]. In Pharmaceutical applications, chitosan is used as a component in hydrogel. The main parameters influencing the characteristics of chitosan are its molecular weight (MW) and its degree of deacetylation [15]. The aim of the present study is the preparation of celecoxib in different chitosan gel formulations and studies of the effect of the concentration of chitosan and its molecular weight on the in vitro release of the drug and its permeation through rat skin. Another aim is to study the performance of the gel formulations on the anti-inflammatory activity of the paw edema in rats.
I. MATERIALS AND METHODS 1. Materials
Celecoxib was kindly donated by Searle, Augusta GA, United States. Chitosan with differing molecular weights came from Fluka Chemie AG (Buchs, Switzerland) and semi-permeable membrane, cellulose in nature with cutoff 1000 Dalton (Cellulose tubing, Sigma Diagnostics, St. Louis, MO, United States). All other chemical were of analytical grade.
2. Preparation of chitosan gels
Chitosan gels were prepared at 1-3 % w/v of the medium molecular weight, 2 % w/v of low, medium and high molecular weight in lactic acid solution (1.0 % v/v). 0.5 % w/v of drug was dissolved in the least amount of propylene glycol and added to the chitosan gel. Methyl paraben sodium salt (0.1 %, w/v) as preservative was added. The contents were stirred and the resulting gel solutions were sonicated to remove the air bubbles. Each of the test samples was transferred into 4-oz tubes till further use.
3. Determination of gel viscosity
Rheological experiments were performed to examine the viscous and elastic properties of the different formulations. Viscosity measurements of gels were performed on a Brookfield Model DV-II+ digital viscometer (Brookfield Engineering Laboratories, INC, Stoughton, United States) at room temperature.
4. In vitro release of celecoxib from gel formulations
In vitro release of the drug from different gel formulations were evaluated using semipermeable membrane as reported by El Maghraby et al. [18]. This employed the FDC-6 Transdermal Diffusion Cell Drive Console (Logan Instrument Corp., NJ, United States). The system is fitted with VTC-200 heater circulator with jacketed vertical glass Franz diffusion cells. The semipermeable cellulose membrane with cutoff 1000 Dalton (Cellulose tubing, Sigma diagnostics, St. Louis, MO, United States) was mounted between the donor and acceptor 201
J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
compartments of the diffusion cell. The receptor compartment was 12 mL and the cells have a diffusional area of 1.7 cm-1. The receptor cell was filled with phosphate buffer (pH 6.8). The system was adjusted to 37 ºC ± 0.5. 0.5 g of the tested formulations was loaded into the donor compartment and occluded using a parafilm. Aliquots (2.0 mL) were withdrawn at specific time intervals and replaced with fresh media. The samples were measured spectrophotometrically (UV-spectrophotometer, Schmidzu-50-02, Kyoto, Japan) at a maximum wave length of 252 nm [19] against similarly treated blanks. The percent cumulative amount of drug released was calculated. All experiments were carried out in triplicate and the average values were calculated.
8. Data analysis
The cumulative amounts of drug permeated with time produced the permeation profiles. These profiles were used to calculate the flux of the drug, which was obtained from the slope of the regression line fitted to the linear portion of the curve. Lag time was calculated from the extrapolation of this line x-axis.
9. Anti-inflammatory activity of the prepared gels
An acute inflammatory activity model, carrageenan-induced rat paw edema method was applied in this study [22, 23]. Measurements of the in vivo anti-inflammatory activity of the formulae conformed to guide lines of the Institutional Animal Ethical Committee of King Saud University. Thirty-six rats weighing about 200 g were divided into six groups of six rats. The animals of group 1 received placebo gel (chitosan gel without drug) and group 2, 3, 4, 5 and 6 received F1 (1.0 % medium molecular weight), F2 (2.0 % medium molecular weight), F3 ( 3.0 % medium molecular weight), F4 ( 2.0 % low molecular weight) and F5 (2.0 % high molecular weight) gel formulations, respectively. Inflammation was produced in the rats using 0.1 mL of 1.0 % w/v carrageenan solution in saline. This was injected subcutaneously into left hind paw. To evaluate the topical anti-inflammatory activity of the gel formulations, paw edema was examined. Thirty minutes later, 0.5 g of each gel was applied topically to the edematous paw. The increase in paw thickness was measured before carrageenan injection (time 0) and 2, 3, 4, 6 and 8 h after carrageenan administration using a dial micrometer. The percentage swelling of the paw and the percent inhibition of edema were calculated. The data were reported as mean ± SD (n = 6).
5. Kinetic treatment of the release data
The in vitro release data of the drug from the investigated gel formulations were studied by curve fitting method to different kinetic models of zero-order, first-order and Higuchi models. - Zero-order release: Mt/Mω = kt
Eq. 1
Ln ( 1- Mt/Mω) = - kt
Eq. 2
(Mt/Mω)2 = kt
Eq. 3
- First- order release:
- Higuchi model :
- The Korsmeyer-Peppas equation : Mt/Mω = ktn
10. Statistical analysis for the obtained results
Statistical analysis for the obtained results was carried out by the student t-test at a 0.05 level of significance.
Eq. 4
This was used to study drug release mechanism by analyzing n as the diffusion exponent. According to this equation, if n ≤ 0.45 a Fickian mechanism, if 0.5 ≤ n ≤ 0.8 a non-Fickian and if 0.8 ≤ n ≤ 1 a zero-order mechanism is governing the drug release mechanism from the gel [20].
II. RESULTS AND DISCUSSION
Chitosan as a non-toxic, biocompatible and biodegradable polymer has been widely used for pharmaceutical and medical application. In this work, Chitosan was used as gel forming agent to study the effect of chitosan concentration and its molecular weight on the release of the drug from the representative formulations. In the preliminary study, the lower molecular weight chitosan required a concentration above 1.7 % (w/v) to produce gel, so we used different concentration of the medium molecular weight polymer. Visual inspection of freshly prepared formulae revealed smooth homogenous topical preparations with acceptable spreadability.
6. Preparation of skin samples
A skin permeation study of celecoxib gel formulations was carried out using full thickness rat skin [21]. To obtain skin, male Wister rats weighing 200-250 g were used in this study. All experimental procedures were in accordance with the guidelines of the Institutional Animal Ethical Committee of King Saud University. Rates were anesthetized using sodium pentobarbitone (60 mg/kg) intraperitoneally. The abdominal skin was shaved using an animal hair clipper, in the direction of the tail to head. The skin was excised from the abdominal region of the sacrificed rats. The dermis part of the skin was wiped three times with a wet cotton swab soaked in isopropanol to remove any adhering fat material. Then, the skin was kept in normal saline solution for two hours. The cleaned skin was washed with distilled water, wrapped in aluminum foil, and stored in a deep freezer at - 20 °C until further use.
1. Viscosity study
It was seen from the flow curves (Figures 1 and 2) that celecoxib containing chitosan gels exhibit psudoplastic flow and the viscosity increases significantly with increasing either the chitosan concentration (Figure 1) or its molecular weight (Figure 2). The viscosity of 3.0 % (w/v) chitosan gel was found to be higher than that of 2.0 % (w/v) and 1.0 % (w/v). Also the viscosity of 2.0 % (w/v) chitosan gel prepared from a high molecular weight was found to be higher than that of medium and low molecular weight (Table I).
7. Skin permeation studies
As mentioned in the in vitro release experiments, skin permeation studies were carried out using an FDC-6 Transdermal Diffusion Cell Drive Console (Logan Instrument Corp., NJ, United States). The skin was mounted with the stratum corneal side uppermost on the vertical glass diffusion cells. The mounted skin was left to equilibrate overnight. 0.5 g of the tested formulations was loaded on the skin surface into the donor compartment and occluded with parafilm. Samples were withdrawn periodically and replaced with fresh medium. These samples were analyzed for drug concentration. All experiments were carried out in triplicate and the average values were calculated.
Table I - Relationship between the viscosities (cP) of the prepared chitosan gel formulations and the percent of celecoxib released after 4 h.
202
Formulation code
Content
Viscosity (cP)
% celocoxib released after 4h
F1 F2 F3 F4 F5
1.0 % MMW 2.0 % MMW 3.0 % MMW 2.0 % LMW 2.0 % HMW
10000 ± 150 13700 ± 220 15250 ± 320 11500 ± 200 17150 ± 250
75 ± 2.2 66 ± 1.8 56 ± 1.5 71 ± 1.5 52 ± 2.2
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
100
60
90 80
% drug released
Shearing rate (s -1)
50 40 30 20 1.0 % (W/V)
10 0 0
500
1000
1500
70 60 50 40 30
2.0 % ( W/V)
20
3.0 % (W/V)
1.0 % (W/V) 2.0 % (W/V)
10
3.0 % (W/V)
2000
2500
0
3000
Shearing stress (dyne/cm2)
100
150
200
250
300
Figure 3 - Release profiles of celecoxib from chitosan gels prepared with different concentration of medium molecular weight chitosan. 100
60
90
% of drug released
50 -1
50
time (min)
Figure 1 - Flow curves of chitosan gels prepared with different concentration of medium molecular weight chitosan.
Shearing rate (s )
0
40 30 20 Low MW 10
Medium MW High MW 500
1000
1500
2000
2500
70 60 50 40 30
Low MW
20
Medium MW High MW
10
0 0
80
0
3000
2
0
50
100
150
200
250
300
time (min)
Shearing strees (dyne/cm ) Figure 2 - Flow curves of chitosan gels prepared with 2.0 % (w/v) of different molecular weight chitosan.
Figure 4 - Release profiles of celecoxib from chitosan gels prepared with 2.0 % (w/v) of different molecular weight chitosan.
2. In vitro drug release study
be related to the drug trapped between the tortuous ways of the gels which take longer to be released [20]. Viscosity of the gel formulation may be a very important factor that affects the release of drug since it may reduce diffusion rate of drug from the vehicles. Hereby, a reverse relationship was observed between the viscosity of chitosan gel and the percent of celecoxib released (Table I). The gel formulation containing 1.0 % (w/v) chitosan with a medium molecular weight has the lowest viscosity (10000 ± 150 cP) and showed the highest percent of drug released (75 ± 2.2). While 2.0 % (w/v) chitosan with a high molecular weight has the highest viscosity (17150 ± 250 cP) showed the lowest percent of drug release (52 ± 2.2).This result was in agreement with El-Maghraby [18], who is reported that the release rate of hydrocortisone is dependent on the viscosity of the microemulsion formulations. A similar finding was obtained by Hirano et al. [24] who concluded that, the higher the viscosity of the gel, the slower the drug released. Ismail et al. [25] observed that the release rate of carbachol chloride from carbopol 934 gel was greater than that from carbopol 940 gel. These results were attributed to the fact that cabopol 934 gel base exhibited a lower viscosity value than that of carbopol 940 gel base and so, it released the drug rapidly [26]. This is in accordance with the data obtained by Eros et al. [27] who studied the effect of viscosity on the drug release from different topical formulations. They found a reciprocal correlation between viscosity and the amount of the drug released. Release kinetic models are shown in Table II. As this table indicates
Drug release study from gel formulations is an important step during the development stages of topical formulation. To study the simultaneous effect of the molecular weight and the concentration of chitosan on the release profiles of drug, the percentage release of the drug from various chitosan gel formulations through a four hour period was calculated (Figure 3). Increasing the chitosan concentration of the medium molecular weight from 1.0 % (w/v) to 3.0 % (w/v) resulted in decreasing the percent of drug released (p < 0.05). With 1.0 % (w/v) gel formulation, 75 ± 2.2 % of celecoxib was released. For 2.0 % (w/v) chitosan gel, the percent release of drug was 66 ± 1.8 %. Increasing the gel forming agent to 3.0 % (w/v), the percentage of the drug was 56 ± 1.5 %. The effect of chitosan molecular weight on the release of drug from gel formulations was studied (Figure 4). The concentration of gel forming agent used was 2.0 %. There is an inversely proportional relationship between the molecular weight of chitosan used and the percent release of drug from prepared gels (p < 0.05). For the high molecular weight grade, the drug release was 52 ± 2.2 %, whereas the drug release was 66 ± 1.8 % and 71 ± 1.5 % for medium and low molecular weight grades. The results of the release test indicate that increasing the chitosan concentration or chitosan molecular weight reduces the release of drug as the penetration rate of water decreases in higher viscosities of the gels. All the drug release curves seem to follow biphasic pattern, with a faster rate of drug release over the first 90 min followed by a slower release for the remaining time that may 203
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Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
the correlation coefficient of the release data fitted to Higuchi model which is higher than other models and all chitosan gel formulations, the diffusion exponent of Peppas equation is less than 0.5 which indicates a Fickian mechanism is dominant and controls the drug release from chitosan gels.
indicates that there was an inversely proportional relationship between the viscosity of the gel formulation and the flux of the drug. Also, the flux of celecoxib of F3 is higher than F4 and F5. Correlation between the skin permeation and the release results showed that the release rate was greater than the permeation flux of drug in all gel formulations. Hillton et al. [29] has reported that there are many factors that may influence the extent of percutaneous absorption of a drug. Partitioning of the chemical between the vehicle and the stratum corneum results in a concentration gradient developing across the skin, influenced by chemical-vehicle-skin interaction.
3. Drug permeation through rat skin
4. Anti-inflammatory activity studies using paw edema
Topical anti-inflammatory activity of semisolid preparations has been reported when applied 1 and 2 h before carrageenan treatment as mentioned by Hiramatsu et al. [30]. Clinically, it seems more reasonable to apply the anti-inflammatory topical preparations after the inflammation stimulus [23]. Table IV illustrates the anti-inflammatory activity of different gel formulations on the hind paw of the rats. It is shown that all the gel formulations have a significant effect (p < 0.05) as anti-inflammatory vehicle, but to a variable extent (inhibition
600
-1
Cumulative amount permeated (ug cm )
-2
Cumulative amount permeated (ug cm )
In vivo measurement of drug administered transdermally is a complex process. Due to the lack of availability of human skin, the in vitro drug permeation through skin was introduced as an alternative way to measure drug permeation. The Wister rat skin model has been used extensively, in view of the fact that its stratum corneum thickness, as well as water permeability, is the same as that of human skin [28]. In this study, drug permeation prior to steady state was similar for all gel formulations during the first hour, as it can be seen in Figures 5 and 6. After that the permeation is changed according to the content of gel formulations. From Table III, it was observed that the lag time for all formulae was between 0.5 and 0.75 h. The flux of celecoxib during the steady state of formula F3 was significantly higher than that of F2 and F1 that according to the viscosity of each formula. This
500 400 300 200 1.0 % (W/V)
100
2.0 % (W/V) 3.0 % (W/V)
0 0
2
4
6
8
10
600
500
400
300
200 Low MW
100
Medium MW High MW
0
0
12
2
4
6
8
10
12
time (hr)
time (hr)
Figure 5 - Permeation profiles of celecoxib obtained after application of chitosan gels prepared with different concentration of medium molecular weight chitosan.
Figure 6 - Permeation profiles of celecoxib obtained after application of chitosan gels prepared with 2.0 % (w/v) of different molecular weight chitosan.
Table II - Kinetic models of celecoxib release from different chitosan gels. Table III - The permeation parameters of celecoxib obtained from different gel formulations.
Gel form
Zero-order (r)
First-order (r)
Higuchi model (r)
n
F1 F2 F3 F4 F5
0.856±0.025 0.900±0.003 0.909±0.010 0.841±0.015 0.925±0.010
0.929±0.015 0.960±0.010 0.950±0.023 0.920±0.025 0.953±0.021
0.980±0.011 0.987±0.002 0.991±0.001 0.984±0.002 0.992±0.004
0.44±0.02 0.45±0.03 0.48±0.01 0.44±0.02 0.45±0.03
Formulation code
Flux (µg cm-2 h-1)
Lag time (h)
F1 F2 F3 F4 F5
35.75 ± 0.35 48.35 ± 2.15 69.38 ± 3.25 34.30 ± 1.12 59.10 ± 2.50
0.75 ± 0.05 0.55 ± 0.03 0.50 ± 0.02 0.71 ± 0.13 0.61 ± 0.09
r: correlation coefficient. n: release exponent of Korsmeyer-Peppas equation. Table IV - Anti-inflammatory activity of celecoxib in different gel formulations. Rat group No.
Gel formulation
1 2 3 4 5 6
control F1 F2 F3 F4 F5
% swelling of induced edema 2h
3h
4h
6h
8h
95 ± 0.5 (5.00) 51.0±0.5 (48.8) 65.1±0.4 (35.9) 61.2±0.8 (39.0) 56.1±0.4 (44.9) 73.2±0.3 (27.8)
94.0 ± 0.5 (6.0) 40.1±0.6 (59.4) 60.2±2.0 (39.9) 54.3±0.5 (45.1) 51.5±2.0 (47.9) 65.3±0.2 (34.5)
93.0 ± 1.0 (7.2) 33.5±0.5 (65.6) 50.2±1.5 (50.2) 51.2±0.7 (49.5) 50.5±1.2 (50.5) 58.4±1.1 (42.3)
91.0 ± 0.6 (9.2) 30.3±0.4 (69.5) 45.3±0.9 (55.5) 45.1±0.5 (54.4) 40.3±1.2 (58.9) 52.6±1.3 (48.5)
90 ± 0.5 (10.0) 21.5±0.3 (79.5) 40.5±2.5 (59.5) 41.4±1.5 (58.6) 32.5±1.1 (67.5) 47.8±0.9 (53.3)
Mean ± SD. The value between parentheses indicates the % inhibition of edema. 204
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J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
percentage about 27.8-79.5) over time course studied (1-8 h) as compared with control. After the first 2 h of the observation, F1 chitosan gel shows 48.8 % inhibition while F5 chitosan gel was 27.8 %. After 6 h, the inhibitory effect was 79.5 and 53.3 for the same chitosan gel forms, respectively. The rank order of edema inhibition using this method was in the following order F1 > F4 > F2 > F3 > F5. This order is similar to that appearing in the percent of drug release. This evident a strong correlation between the percent of drug release from the various chitosan gel formulations and the anti-inflammatory activity. Correlating the in vitro release results (Table I), permeation results (Table III) and anti-inflammatory activity (Table IV). The formulation were ranked in the term of release rate and anti-inflammatory activity as F1 > F4 > F2 > F3 > F5. In terms of skin permeation they were ranked as F3 > F5 > F2 > F1 > F4. This evident taking into consideration that the release rates of drug were greater than the transdermal flux in all cases. In addition, there were no significant differences between the lag time values obtained after application of different formulations. The lag time is a permeation parameter depending mainly on the diffusivity of the drug through the skin. In this study, by increasing the permeation of drug through the skin, the localization of drug in epidermal layer decreases so the anti-inflammatory activity decreases and vice versa.
10. 11.
12. 13. 14.
15. 16.
*
17.
The results of in vitro release, permeation and anti-inflammatory studies concluded that, a chitosan is a good carrier for celecoxib as gel formulation. The polymer concentration and the molecular weight of chitosan are very effective factors on the release of drug from gel formulations. Celecoxib permeation through rat skin is independent of the viscosity of the gel. All gel formulations have significant antiinflammatory activity.
18. 19. 20. 21.
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J. DRUG DEL. SCI. TECH., 21 (2) 201-206 2011
Preparation, characterization and anti-inflammatory activity of celecoxib chitosan gel formulations M. El-Badry, G. Fetih
AcknowledgmentS
Manuscript
The authors would like to thank Dr. Mohamed A. Ibrahim for his advices during rewriting of the manuscript. Also, we would like to appreciate Dr. M. Kazie for revision the manuscript.
Received 6 December 2010, accepted for publication 14 March 2011.
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