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Optimization and formulation design of carbopol loaded Piroxicam gel using novel penetration enhancers
International Journal of Biological Macromolecules 55 (2013) 246–253
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Optimization and formulation design of carbopol loaded Piroxicam gel using novel penetration enhancers Hema Chaudhary ∗ , Ajay Rohilla, Permender Rathee, Vikash Kumar P.D.M. College of Pharmacy, Sector: 3A, Sarai Aurangabad, Bahadurgarh, Haryana, India
a r t i c l e
i n f o
Article history: Received 6 December 2012 Received in revised form 8 January 2013 Accepted 14 January 2013 Available online 31 January 2013 Keywords: Formulation Drug transport Drug design Box–Behnken design Drug delivery
a b s t r a c t The aim of the study was to develop and optimize Piroxicam transdermal gel formulation using threefactor, three-level Box–Behnken design by deriving a second-order polynomial equation to construct contour plots for prediction of responses as three selected independent variables with ratio of carbopol 974 (X1 ), ratio of propylene glycol (PG) (X2 ) and ratio of ethanol (X3 ). The dependent variables studied were the skin permeation rate of piroxicam (Y1 ), viscosity of the gel (Y2 ) and pH of the gel (Y3 ). Response surface plots were drawn, statistical validity of the polynomials was established to find the compositions of optimized formulation which was evaluated using the vertical Franz-type diffusion cell. The permeation rate of piroxicam increased proportionally with ethanol concentration but decreased with polymer concentration. The design demonstrated the role of the derived polynomial equation and contour plots in predicting the values of dependent variables for the preparation and optimization of gel formulation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Today about 74% of drugs are taken orally and are found not to be as effective as desired. Discovering a new medicine is a very expensive and time consuming process that makes re-designing the modules a more lucrative task. The design of dosage form, whether a tablet, an injection or a patch is to deliver the right amount of medicine at right time to the right target site and it becomes complicated if each medication were to be delivered in an optimal and preferred manner to the individual patient. The medication may not be absorbed if it is released too slowly or if it is delivered too rapidly, the patient may suffer untoward effects and its desired effects may not last as long as needed. Management of illness through various conventional systems of medications involves various problems and complications such as attainment and maintenance of drug concentration in the body within a therapeutic range. It is often necessary to take it several times a day so as to prevent the concentration dropping below the minimum effective level and to exhibit best therapeutic effects. A continuous intravenous infusion is considered as a superior mode of drug administration as compared to oral route. However, to overcome the problems associated with intravenous infusion and to duplicate closely its benefits, skin is used as a most favorable part of drug administration. So, whilst skin is used as a part of drug administration, it is foretold as transdermal administration
∗ Corresponding author. Tel.: +91 8901451603. E-mail address: [email protected] (H. Chaudhary). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.01.015
and the drug delivery through transdermal administration is called as transdermal drug delivery system (TDDS). TDDS is a promising method of drug administration that can avoid the variability in rates of absorption and metabolism encountered in oral treatment [1,2]. Drug Delivery via. the transdermal route is an interesting option because transdermal route is convenient and safe. The positive features of delivering drugs across the skin is to achieve systemic effects by avoidance of first pass metabolism and gastro intestinal incompatibility as it helps to predict and extend duration of activity. TDDS is also used to minimizing undesirable side effects and provides utilization of drugs with short biological half lives with narrow therapeutic window by improving physiological and pharmacological response. This system avoids the fluctuation in drug levels with maintenance of plasma concentration of potent drugs and provides suitability for self-administration and enhanced therapeutic efficacy [3]. There are three approaches for permeation enhancement; drug vehicle based enhancement; physical and chemical enhancement. Chemical enhancement is affected by penetration enhancers [4–7]. They are used for improving transdermal drug delivery by reversibly reducing the barrier resistance. Their mechanisms include disruption of intercellular lipid and/or keratin domains and tight junctions which results in enhanced drug partitioning into tissue or by altering thermodynamic activity/solubility of drug. There are numerous other chemicals that are able to exert the above mentioned effects such as azone, urea, alcohols, surfactants, sulphoxides, glycols and fatty acids. It has been proposed that any chemical that is used in facilitation of transdermal drug delivery should ideally be non-toxic and non-irritating to skin. It should
H. Chaudhary et al. / International Journal of Biological Macromolecules 55 (2013) 246–253
have solubility factor similar to skin and when removed the barrier properties of the skin should reform fully and rapidly. It should be cosmetically acceptable, compatible with wide range of excipients and pharmacologically and chemically inert with no pharmacological action in the body. The terpenes are usually the constituents of volatile oil and categorized in GRAS category by USFDA having pharmacologically inert, non-toxic and non-irritating effect to the skin [8–14]. Terpenes are secondary metabolites that are mostly synthesized in plants. They are found in essential oils consisting of five carbon isoprene unit which undergoes a condensation reaction to forms a much larger (10–40 carbon) terpene frame work. This larger terpene structure can be subjected to further biochemical processing, which produces final range of terpenes. Thousands of different terpenes (monoterepenes, sesquiterpenes and diterpenes) have been identified till date and many have been investigated as penetration enhancers due to their high lipophilicity. They have good safety profile and are non-toxic and non-irritant to the skin. They also possess attributes such as low melting point and low molecular weight. They have high percutaneous enhancement ability, reversible effect on the lipids of stratum corneum, minimal percutaneous irritancy at low concentrations (1–5%). The mechanism of action of terpenes involves increasing one or more of the following effects: diffusion coefficient, partition coefficient, drug solubility (increasing the thermodynamic activity of the drug), lipid extraction (i.e. disruption of lipid–protein domain), macroscopic barrier perturbation and molecular orientation of terpenes molecule within the lipid bilayer. However, the rate and extent of enhancement are dependent upon type and physicochemical characteristics (such as melting point, solubility) of terpene, concentration of terpene used, absence or presence of co solvent or enhancer and concentration of co-solvent or enhancer if present. In recent years, many attempts have been made to investigate the use of terpenes as skin permeation enhancers including menthol, linalool, limonene and carvacrol to promote the transdermal transport of drugs including chiral agents. Monoterpenes were selected due to their small sizes which promote its good penetration enhancer capability. The objective of the study was to develop a low dose transdermal delivery system i.e. transdermal carbopol loaded gels for piroxicam that would provide the release of drug at a controlled rate to achieve a therapeutic drug level for a prolonged period by using myrcene and 3-Carene (monoterpene hydrocarbon are selected since they are lipophilic and smaller in size and a novel approach) as chemical penetration enhancers for the efficacious transdermal delivery of piroxicam.
2. Materials and methods Piroxicam pure was purchased from Taj Pharmaceuticals Limited (India). Carbopol (CP-974) was obtained from Central drug house (Delhi, India). Penetration enhancers (3-Carene and myrcene) were purchased from Sigma Aldrich Chemicals Pvt. Ltd. (UK). Ethanol was purchased from Jiangsu Huaxi International (China). Propylene glycol (PG), methyl and ethyl paraben sodium and sodium hydroxide (pellets) were bought from Central drug house (Delhi, India). All the materials used in the study were of analytical grade and double distilled water was used throughout the study.
2.1. Preliminary trials The experimental field should neither be too large nor too small, leading to nonrealistic experiments from an optimal region [15].
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2.1.1. Selection of polymer For the preparation of gel various polymers were used such as HPMC, carbopol, carboxymethyl cellulose, sodium carboxymethyl cellulose, carboxymethyl mungbean starch, chitosan, etc. with different grades. The carbopol on the basis of its good consistency was used. Many dummy gels were prepared with this polymer at different concentration and the concentrations between 1% and 2% gave good consistency for the gel and it is generally recognized as safe (GRAS) property. Thus, carbopol 974 in the concentration range of 1–2% (w/w) was selected for gel preparation. 2.1.2. Optimization of enhancer Monoterpenes such as myrcene and 3-Carene were selected due to their small size which promotes its good penetration enhancer capability. The working concentration range of the both the enhancer was determined by ex vivo permeation study. The flux was determined from Fick’s law of diffusion considering the transport of drugs across the skin barrier as a process of passive diffusion. The cumulative drug release was calculated as concentration in total volume divided by skin surface area. The flux (g/cm2 /h) was calculated from the slope of the linear portion of the cumulative amount permeated per unit area versus time plot. After ex vivo skin permeation study of piroxicam with different concentrations of both the enhancers, the enhancer was optimized on the basis of cumulative amount of drug release and flux. The steady state permeability coefficient (Kp) and enhancement ratio (ER) were calculated by using following equation: Kp =
J C
where J is the flux and C is the initial concentration of piroxicam. ER = flux of piroxicam containing chemical penetration enhancer/flux of piroxicam without using enhancer. 2.2. Preparation of gels Gels were prepared by dispersing the polymer (Carbopol 974) and slowly added into known quantity of water under constant stirring with addition of methyl paraben sodium (0.01%, w/v) and propyl paraben sodium (0.1%, w/v) and kept for a period of 24 h for soaking. The dispersion was then allowed to hydrate and swell for 60 min. After hydration, drug (0.5%, w/w) was dissolved in appropriate quantity of propylene glycol, ethanol and 12.5% of optimized penetration enhancer i.e. 3-Carene with constant stirring and blend was transferred to container containing soaked carbopol and agitated for additional 20 min. Tri-ethanol amine (TEA) was added in the dispersion and stirred thoroughly until clear viscous homogeneous gel was obtained and pH was kept in the range of 6.0–7.0 for maximum efficiency. The formulations were prepared incorporating CP-974 (1–2%, w/w), PG (5–15%, w/w), ethanol (10–30%, w/w), penetration enhancer (12.5%, v/w) and water using Box–Behnken experimental design and optimized formulation was generated using statistical screening. 2.3. pH evaluation A defined amount of Piroxicam gel (100 mg) was measured and diluted with distilled water and mixed well. The pH of the gel was recorded using Digital pH Meter (Multitech Instrument Co. Pvt. Ltd., Delhi, India) by bringing it in contact with the gel and allowing it to equilibrate for one min. pH evaluation was carried out for all experimental formulations. Experiments were performed in triplicate to check for the neutralization of gels of different batches.
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H. Chaudhary et al. / International Journal of Biological Macromolecules 55 (2013) 246–253 Table 1 Variables with coded values in statistical design.
2.4. Rheological measurements The viscosity measurement of gel was measured by Brookfield viscometer DV-I at 10 rpm (DVS-I Prime, India). Samples of the gels were allowed to settle over 30 min at 25 ± 1 ◦ C before measurements were taken. All viscosity measurements were performed in triplicate. 2.5. Spreadability Spreadability was determined on the basis of slip and drag character of gels after 72 h of preparation. The gel were filled in collapsible tubes and sealed by crimping one of the ends. The weights of the tubes were recorded. The tubes were placed between two glass slides and were clamped. Certain load of weight based on quantity of gel was placed over the slides and then cap was removed. The amount of extruded gel was collected and weighed. The percentage of gel collected was calculated [16]. Spreadability is expressed in terms of time in seconds to slip off from gel and placed in between slides under the direction of certain load. Lesser the time taken for separation of two slides, better the spreadability. It is calculated by using the formula: S=
M×L T
where S represents the spreadability (g/s), M is the weight in pan (g), L is the fixed distance moved by the slide and T is the time. 2.6. Experimental design The formulations were optimization using 3-factor, 3-level factorial design by constructing second order polynomial models and exploring quadratic response surfaces with Design Expert® (version 8.0.7.1, Stat-Ease Inc., Minneapolis, Minnesota). The selected independent and dependent variable with coded levels, low, medium and high levels are listed in Table 1. The experimental design generated polynomial equation, is given as Y0 = b0 + b1 X1 + b2 X2 + b3 X3 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + b11 X12 + b22 X22 + b33 X32 where Y0 is the dependent variable; b0 is the intercept; b1 to b33 are the regression coefficients computed from the observed experimental values of Y; and X1 , X2 and X3 are the coded levels of independent variables. The terms X1 , X2 and Xi2 (i = 1, 2, or 3)
Coded levels
Independent variables in % w/w
Low (−1) Medium (0) High (+1)
X1 1 1.5 2
X2 5 10 15
X3 10 20 30
Dependent variables
Constraints
Y1 = flux of piroxicam (g/cm2 /h) Y2 = viscosity of gel (cP) Y3 = pH of gels
32 ≤ Y1 ≥ 110 1182 ≤ Y3 ≥ 3956 6.09 ≤ Y1 ≥ 6.7
X1 = polymer conc. (%); X2 = PG conc. (%); X3 = ethanol conc. (%).
represent the interaction and quadratic terms, respectively [17]. The amount of polymer concentration (X1 ), PG (X2 ) and ethanol (X3 ) used to prepare each of the formulations and their observed and predicted responses are given in Table 2. 2.7. In vitro drug release studies 2.7.1. Preparation of rat skin The animals used for preparation of skin were male albino rats weighing 110–125 g (excised from the abdominal region). The rats were sacrificed by giving excess anesthesia (ether). On the day of experiment the skin was thawed to room temperature and hair and fat were removed. The hair on the abdominal region was carefully removed using hair removal cream and hair removal spatula and adhering subcutaneous fat was cleaned using isopropyl alcohol (IPA) by rubbing with cotton previously dipped in IPA and immediately kept in isotonic phosphate buffer (IPB) solution. This step caused the layer to remain unwrinkled, and then the skin was washed with distilled water [18]. 2.7.2. Permeation studies Ex vivo permeation studies were carried out through rat skin using an automated transdermal Franz vertical diffusion cell sampling system (Rama Glasswares Co. Pvt. Ltd., Delhi, India). The skin was trimmed to the appropriate size and mounted on the diffusion cell such that the stratum corneum of the skin faced the donor cell and dermis faced the receiver cell. The cell was mounted using spring. The receptor compartment was filled with the vehicle (i.e. PEG 400:IPB pH 7.4 (30:70) and the donor compartment was filled with 5 mg drug suspension (0.5%, w/v) containing different concentration of chemical penetration enhancer. The whole assembly was
Table 2 Design of experiments with results. Runs
Batches
X1
X2
X3
Y1 Y2 (Experimental)
Y3
Y1 (Predicted)
Y2
Y3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
GF1 GF2 GF3 GF4 GF5 GF6 GF7 GF8 GF9 GF10 GF11 GF12 GF13 GF14 GF15 GF16 GF17
0 −1 −1 −1 +1 0 0 −1 0 0 0 +1 +1 +1 0 0 0
+1 0 −1 0 0 +1 0 +1 −1 −1 0 0 −1 +1 0 0 0
−1 −1 0 +1 +1 +1 0 0 −1 +1 0 −1 0 0 0 0 0
68.2 99.9 110.5 85.8 21.2 36.1 72.3 102.2 40.8 48.7 75.2 31.9 29.5 52.7 75.1 74.2 76.2
6.29 6.16 6.31 6.09 6.50 6.30 6.30 6.10 6.50 6.50 6.30 6.51 6.70 6.50 6.20 6.30 6.20
68.3 100.0 110.7 85.8 21.0 36.1 72.6 102 40.7 48.5 75.2 32.0 30.2 52.7 75.1 74.2 76.2
3017.1 2338.7 2821.3 2927.2 4248.0 3847.9 3446.1 2756.5 3117.2 3955.9 3488.6 1182.0 3951.9 3808.0 3454.2 3466.1 3459.8
6.30 6.20 6.30 6.09 6.55 6.29 6.20 6.15 6.46 6.47 6.30 6.51 6.70 6.49 6.20 6.30 6.20
3016.5 2338.5 2821.1 2926.8 4248.5 3848.1 3445.7 2756.1 3117.0 3956.1 3489.0 1182.4 3951.3 3807.7 3453.7 3465.7 3460.0
X1 = polymer conc.(%); X2 = PG conc. (%); X3 = ethanol conc.(%); Y1 = flux of Piroxicam gel (g/cm2 /h); viscosity of gel (cP); pH of gels.
All batches of formulated gel were characterized for their cosmetic persona (color, scent, texture and consistency). The formulated gels were light yellowish in color with a pleasurable, smooth homogeneous appearance and texture. The pH value of all the prepared formulations ranged from 6.09 to 6.7 as shown in Table 2. The spreadability plays a considerable role in patient fulfillment and helps in uniform application of gel to the skin. A good gel takes less time to spread and will have high spreadability. The spreadability of formulated gels was decreased as the concentration of polymer increased. The values of spreadability
59.3 73.7 120.4 205.2 307.7 530.4 614.8 778.5 955.5 1208.3 1427.1 1721.4 74.2 0.015 4.22 1.90
GF17 GF16 GF15
60.7 74.7 121.5 206.1 310.3 530.8 619.0 787.4 957.3 1221.9 1429.5 1729.2 75.1 0.015 4.16 1.80 60.5 73.1 146.2 182.2 250.5 409.7 462.3 581.6 630.4 832.8 1048.7 1261.5 52.7 0.011 5.10 2.74
GF14 GF13
0.80 3.84 15.8 40.1 63.2 106.3 150.7 207.9 260.3 396.6 543.2 693.5 29.5 0.006 1.83 3.56 3.03 6.47 19.8 35.2 39.8 62.6 105.3 172.5 223.9 405.3 542.7 721.3 31.9 0.006 3.31 3.50
GF12 GF11
61.9 76.7 123.5 209.5 313.8 536.0 622.3 792.3 957.9 1226.8 1432.4 1734.1 75.2 0.015 4.16 1.73 117.6 184.2 217.4 285.2 329.6 503.2 639.1 719.0 821.4 992.5 1102.4 1209.9 48.7 0.010 3.12 2.91
GF10 GF8 GF7 GF6 GF5 GF4 GF3 Formulation code
3.2. Characterization of gels
Cumulative amount of drug release (g/cm2 )
The ex vivo skin permeation study of piroxicam was performed with different concentration of enhancers. Myrcene at different concentrations such as 5%, 10%, 15% (w/w) did not give good satisfactory results as shown in Table 3. Further, 3-Carene at concentration of 5%, 10%, 15% (w/w) showed that the flux increased in increasing order of enhancer concentration and decreased at 15% (w/w) concentration. The reason of decreased release of drug was due to attainment of saturation point. Thus, the concentration of 12.5% of 3-Carene was selected for further study due to the improved flux (89.26 g/cm2 /h). The satisfactory flux was also not obtained when the mixture of myrcene and 3-Carene were tried which may be due to immiscibility of these enhancers.
Time (h)
3.1. Optimization of enhancers
Table 4 Ex vivo skin permeation study of drug-carbopol loaded gel with optimized penetration enhancer.
3. Result and discussion
GF9
The statistical polynomial equation generated by Design Expert® (version 8.0.7.1, Stat-Ease Inc., Minneapolis, Minnesota) was established on the basis of ANOVA terms in the software. The totals of 17 runs (GF1–GF17) with triplicate center points were generated. The models were evaluated in terms of statistically significant coefficients and R2 values. The compositions of optimized formulation over the whole experimental region were found by conducted validation of RSM results.
4.85 11.3 22.9 36.2 49.6 88.3 168.4 263.6 466.8 708.5 829.5 1054.7 40.8 0.008 3.24 3.10
2.8. Response analysis for optimization
33.2 102.8 145.5 283.6 423.5 573.9 810.3 924.1 1119.8 1500.1 1952.3 3102.5 102.2 0.020 5.36 1.20
placed on magnetic stirrer and stirred magnetically using magnetic bead at 600 rpm placed in receiver cell. Drug permeation studies were carried out for 24 h at a temperature of 37 ± 2 ◦ C. The aliquots of 0.5 ml were withdrawn from the receptor compartment at predetermined time intervals of 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 h and were replaced by the same volume of PEG 400:IPB pH 7.4 (30:70). Receiver volume was immediately replenished with the same volume of fresh vehicle. Permeation parameters like flux (g/cm2 /h) and enhancement ratio were calculated.
58.3 69.0 117.2 200.8 302.9 529.8 607.9 765.0 905.1 1189.9 1350.2 1707.9 72.3 0.014 4.23 2.12
– 2.28 2.36 2.45 2.85 4.47 4.55 4.08 1.33
141.7 189.7 201.6 244.5 279.1 329.9 428.7 466.6 544.1 640.5 724.9 798.4 36.1 0.007 2.88 3.40
0.0039 0.0089 0.0092 0.0095 0.0111 0.0174 0.0178 0.0159 0.0052
22.3 39.5 56.3 66.8 82.8 137.2 147.8 180.2 205.3 317.8 450.4 541.3 21.2 0.004 1.82 3.72
19.59 44.73 46.37 48.09 56.03 87.67 89.26 79.98 26.17
15.2 37.7 83.0 110.9 183.2 288.5 373.9 651.2 724.5 1162 1554.7 1925.1 85.8 0.017 4.81 1.40
Piroxicam (control) Piroxicam + myrcene (5%) Piroxicam + myrcene (10%) Piroxicam + myrcene (15%) Piroxicam + 3-Carene (5%) Piroxicam + 3-Carene (10%) Piroxicam + 3-Carene (12.5%) Piroxicam + 3-Carene (15%) Piroxicam + myrcene (5%) + 3-Carene (5%)
100.8 157.7 297.6 368.2 443.5 630.8 882.2 1076.5 1302.8 1495.8 1821.8 3387 110.5 0.022 5.39 1.00
Enhancement ratio
92.7 115.9 150.7 284.1 355.3 526.9 767.3 841.7 947.3 1265.6 1714.5 1995.5 99.9 0.019 5.16 1.50
Permeability coefficient (Kp)
GF2
Flux (g/cm2 /h)
GF1
Formulation
0.5 33.2 53.0 1.0 2.0 72.1 3.0 125.5 4.0 139.1 6.0 258.7 356.7 8.0 422.9 10.0 12.0 671.3 16.0 890.5 20.0 1105.4 24.0 1508.5 Flux (g/cm2 /h) 68.2 Permeability 0.014 Coefficient enhancement ratio 5.03 2.40 Retained concentration (g/ml)
Table 3 Ex vivo study of piroxicam with chemical penetration enhancer.
64.8 80.2 125.7 220.8 319.1 547.8 636.0 810.9 984.2 1248.5 1438.5 1746.8 76.2 0.015 4.10 1.64
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Table 5 Summary of results of quadratic model for regression analysis of responses Y1 , Y2 , and Y3 . Quadratic model
R2
Adjusted R2
Predicted R2
SD
%CV
Response (Y1 ) Response (Y2 ) Response (Y3 )
0.9992 0.9999 0.9689
0.9982 0.9998 0.9289
0.9980 0.9998 0.8964
1.51 12.62 0.040
1.78 0.04 0.70
[25.83–33.85 (g cm)/s] indicate that the gels were easily spreadable by small amount of shear and possessed acceptable bioadhesion. 3.3. Rheological measurements The viscosity of all the 17 formulations ranged between 1182 and 3951 cP as shown in Table 2. 3.4. Evaluation of in vitro release The effect of optimized penetration enhancer on permeation of piroxicam was evaluated for cumulative amount of drug release. The result of skin permeation studies of drug loaded gel is shown in Table 4. It was found that among all the formulations, GF3 showed highest cumulative amount of drug release (3387.04 g/cm2 ), flux (110.53 g/cm2 /h), permeability coefficient (0.0221) and there is 5.39 folds increase in flux as compared to control gel which is due to reduced inter particular cross linking between drug and polymer causing increase in rate of drug release. The permeation of drug was increasing by using of optimized penetration enhancer i.e. 3Carene as it has potential to solubilize the lipids in stratum corneum without damaging the skin and this leads to enhancement of drug permeation. 3.4.1. Histopathology study A comparative study of penetration enhancing capacity of an optimized formulation gel (GF3) was performed and further compared with the untreated rat skin as shown in Fig. 1. The rat skin section treated with an optimized formulation gel (GF3) showed a significant increase of disruption of stratum corneum organization. The rat skin treated with an optimized formulation gel (GF3) in Fig. 1b showing enlarged intercellular space along with clear reversible disruption of stratum corneum organization i.e. lipid domains, which leads decrease in degree of lipid arrangement in the stratum corneum confirming the penetration enhancement of an optimized formulation. 3.5. Model fitting to the data The total 17 runs with triplicate center points were generated with the help of three factors, three-level Box–Behnken statistical experimental design and responses are observed. The ranges of Y1 is 1–2%, Y2 is 5–10%, and Y3 is 10–30% for all batches and responses flux of gel, viscosity of gel and pH of gel were found to be in the range 32–110 g/cm2 /h, 1182–3956 cP and 6.09–6.7 respectively. The best-fitted model was quadratic and the comparative values of R2 , SD, and %CV are given in Table 5 along with the regression equation generated for each response. All statistically significant (p < 0.05) coefficients are included in the equations. A positive value shows an effect that favors the optimization, while a negative value shows an opposite relationship between the factor and the response. The three independent variables, namely the concentration of CP-974 (X1 ), PG (X2 ), and ethanol (X3 ) has interactive effects on the three responses (Y1 , Y2 and Y3 ).
Fig. 1. High power photomicrograph of section of rat skin (a) untreated; (b) treated with an optimized formulation (GF3).
3.6. Two dimensional plots and response analysis by polynomial equations Two dimensional plots (contour plots) were prepared for all the three responses as shown in Figs. 2–4 for responses Y1 , Y2 and Y3 , respectively. These plots showed the interaction effects of the factors which are useful in studying the effects of two factors on each response at a particular point. 3.6.1. Effect on flux: response 1 (Y1 ) The following polynomial equation was generated for flux of piroxicam Y1 = 74.61 − 32.89X1 + 3.72X2 − 6.10X3 + 7.89X1 X2 + 0.83X1 X3 − 9.99X2 X3 + 5.19(X1 )2 − 6.06(X2 )2 − 20.12(X3 )2 where Y1 is the flux of piroxicam, X1 , X2 and X3 are the concentration of polymer, PG and ethanol respectively. The model F-value of 981.26 implies the model is significant. The lack of fit F-value 95.60 implies the lack of fit is not significant (p ≥ 0.0001). In this case X1 , X2 , X3 , X1 X2 , X2 X3 , X12 , X22 , X32 had a more pronounced effect on flux of piroxicam. The predicted R2 of 0.9980 is in reasonable agreement with the adjustable R2 of 0.9982. The ratio of 101.175 indicates an adequate signal. This model can be used to navigate the design space. The contour plots which show the effect of different independent variables on flux of piroxicam (Y1 ). The amount of polymer increasing will decrease the size of the channels, as does an increase in swelling degree decreases the
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Fig. 2. Contour plot showing the effect of polymer concentration (a) (X1 ) and PG (X2 ); (b) PG (X2 ) and ethanol (X3 ) on response Y1 .
permeation of drug [19]. In our study, it is revealed that flux of piroxicam decreased with increase in concentration of the polymer (CP-974) as the drug remains entrapped in the gel complex of the polymer. The amount of ethanol was not increased beyond 20% as it yielded a hazy product. Flux was found to decrease on increasing the concentration of CP-974 due to increase in viscosity of the gel. A linear relationship was obtained between % (w/w) of PG and flux due to improved drug diffusion as shown in Fig. 2 and flux increased with increase in concentration of ethanol. 3.6.2. Effect on viscosity of gel: response 2 (Y2 ) The following polynomial equation was proposed by the model for viscosity of gel formulation: Y2 = 3462.83 + 543.42X1 − 52.17X2 + 415.61X3 − 19.64X1 X2 + 1119.47X1 X3 − 1.85X2 X3 − 719.59(X1 )2 + 590.81(X2 )2 − 569.17(X3 )2 where Y2 is the viscosity of the gel formulations, X1 is the polymer concentration, X2 is the concentration of PG, and X3 is the concentration of ethanol. The independent factors, polymer concentration is observed to have significantly higher positive effect on the viscosity of the gel due to greater inter particular cross linking between drug and polymer. An inverse relationship was obtained between PG and viscosity due to more spreadability attributed by PG. The viscosity also decreased with increase in concentration of ethanol due to conformational changes in the polymer making the gel. Overall, the model is significant (F-value = 9529.58; p > 0.0001) while the lack of fit is not significant (F-value of 0.08; p = 0.9661). The values for predicted (0.9998) and adjusted (0.9998) R2 values are in reasonable agreement. The signal to noise ratio was found to be satisfactory as the observed adequate precision ratio of 343.618 is above 4. The effect of two independent factors on viscosity at a time as shown in the contour plots (Fig. 3). 3.6.3. Effect on pH of gel: response 3 (Y3 ) The response Y3 relating to pH of gel is shown as: Y3 = 6.26 + 0.19X1 − 0.096X2 − 8.75X3 + 0.015X1 X3 − 2.50X2 X3 + 0.039(X1 )2 + 0.10(X2 )2 + 0.016(X3 )2
where Y2 is the viscosity of the gel formulations, X1 is the polymer concentration, X2 is the concentration of PG, and X3 is the concentration of ethanol. The positive effect of ethanol is found to be more than that of PG on pH. PG does not having any effect on pH of gel but the pH increase with increase in concentration of ethanol as shown Fig. 4. The model F-value of 24.22 implies the model is significant (p > 0.0002) but the lack of fit is not significant. The predicted R2 of response Y3 is 0.8964 which is reasonable agreement with the R2 of 0.9689. The observed adequate ratio was found to be 17.405 is above 4. 3.7. Optimization and data analysis On the basis of physical evaluation and ex vivo skin permeation studies of all control and sample Piroxicam gel formulations, it was found that formulation GF3, GF4 and GF8 gave good results and among these GF3 gave best result. Therefore GF3 was selected as optimized formulation. The optimized formulation of Piroxicam gel (GF3) was compared with marketed formulation for ex vivo skin permeation study as shown in Table 6. The optimized formulation (GF3) showed maximum cumulative amount drug Table 6 Comparative ex vivo skin permeation study of optimized formulation GF3 and marketed formulation of Piroxicam gel. Time (h)
0.5 1 2 3 4 6 8 10 12 16 20 24 Flux (g/cm2 /h) Permeability coefficient (Kp) Enhancement ratio
Cumulative amount of drug permeated (g/cm2 ) GF3
Marketed
100.8 157.7 297.6 368.2 443.5 630.8 882.2 1076.5 1302.8 1495.7 1821.9 3387.0 110.5 0.022 1.67
50.20 60.52 77.73 131.6 144.5 267.8 373.7 433.4 678.7 914.6 1123.1 1490.7 66.10 0.013
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Fig. 3. Contour plots showing the effect of polymer concentration (a) (X1 ) and PG (X2 ); (b) (X1 ) and ethanol (X3 ); (c) PG (X2 ) and ethanol (X3 ) on response Y2 .
release (3387.04 g/cm2 ; flux 110.5 g/cm2 /h) in comparison to marketed formulation (1490.68 g/cm2 ; flux 66.10 g/cm2 /h) with an enhancement ratio of 1.67. 4. Stability studies The accelerated stability analysis for optimized formulation was conducted according to International Conference on Harmonization (ICH) guidelines [20–24]. The optimized gel formulation was stored in well closed glass containers for a period of 90 days at 40 ◦ C temperature and 75% relative humidity in humidity chamber. At predetermined intervals; 0, 30, 60 and 90 days, samples were collected and their physical evaluation parameters such as color, presence of clogs, consistency, phase separation and chemical evaluation parameters such as pH and drug content were evaluated. 5. Conclusion
Fig. 4. Contour plots showing the effect of PG (X1 ) and ethanol (X3 ) on response Y3 .
The Piroxicam gel showed promising results as using carbopol release controlled polymer and optimized using 3-factor, 3-level Box– Behnken design. The quantitative responses; flux of
H. Chaudhary et al. / International Journal of Biological Macromolecules 55 (2013) 246–253
piroxicam, viscosity of gel, and pH of gel for different combinations of independent variables; polymer, PG and ethanol were obtained experimentally and the results were found to fit the design model. The quantitative effect of these factors at different levels on the responses could be predicted by using polynomial. The formulation (GF3), having polymer concentration 1%, PG 5%; ethanol 20%; the flux for formulation was 110.53 (g/cm2 /h) and 2821.15 cP viscosity was chosen as optimized transdermal gel (GF3). Therefore it can be concluded that a transdermal gel formulation for piroxicam was developed and optimized using a 3-factor, 3-level Box–Behnken design. The stability study as per ICH guidelines revealed that the optimized formulation gel (GF3) holds promise for a high stability. Acknowledgements The authors wishes to thank Management of PDM Religious and Educational Association (PDMREA) and also grateful to PDM College of Pharmacy, Sarai Aurangabad, Bahadurgarh, Haryana, India for financial support, and providing the facilities to carry out the research work. References [1] L.V. Allen, N.G. Popovich, H.C. Ansel, Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th ed., Wolter Kluwer Publishers, New Delhi, 2005, pp. 298–299. [2] R. Kumar, A. Philip, Tropical Journal of Pharmacy Research 6 (1) (2007) 633–644. [3] P. Kumar, C. Sankar, B. Mishra, The Indian Pharmacist 5 (3) (2004) 7–17. [4] P.F. Uzor, C.J. Mbah, E.O. Omeje, Journal of Chemical and Pharmaceutical Research 3 (3) (2011) 680–700. [5] B.W. Barry, European Journal of Pharmaceutical Sciences 14 (2001) 101–114.
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[6] D.I.J. Morrow, P.A. McCarron, A.D. Woolfson, R.F. Donnelly, Open Drug Delivery Journal 1 (2007) 36–59. [7] J.E. Seto, B.E. Polat, B. VanVeller, R.F.V. Lopez, R. Langer, D. Blankschtein, Journal of Controlled Release 152 (2011) 1–8. [8] B. Sapra, S. Jain, A.K. Tiwary, American Association of Pharmaceutical Scientists 10 (1) (2008) 120–132. [9] V.R. Sinha, M.P. Kaur, Drug Development and Industrial Pharmacy 26 (11) (2000) 1131–1140. [10] M. Aqil, A. Ahad, Y. Sultana, A. Ali, Drug Discovery Today 12 (2007) 1061–1067. [11] S. Zwenger, C. Basu, Biotechnology and Molecular Biology Reviews 3 (1) (2008) 1–7. [12] T. Ghafourian, P. Zandasrar, H. Hamishekar, A. Nokhodchi, Journal of Controlled Release 99 (2004) 113–125. [13] H.K. Vaddia, P.C. Hoa, Y.W. Chanb, S.Y. Chana, Journal of Controlled Release 81 (2002) 121–133. [14] D. Lahora, V. Chaudhary, S.K. Shah, G. Swami, G. Chaudhary, S.A. Saraf, International Journal of Pharmaceutical Research and Development 2 (12) (2005) 39–45. [15] G.G. Agyralides, P.P. Dallas, D.M. Rekkas, International Journal of Pharmaceutics 281 (1–2) (2004) 35–43. [16] M.N. Mutimier, C. Reffkin, J.A. Hill, E. Marry, M.E. Glickman, G.N. Cry, Journal of American Pharmacists Association 45 (1965) 212–220. [17] G.E.P Box, D.W. Behnken, Technometrics 2 (1960) 455–475. [18] Y.S. Krishnaiah, V. Satyanarayana, P. Bhasker, Drug Development and Industrial Pharmacy 29 (2) (2003) 191–202. [19] J.R. Ebdon, M.S. Jones, Flame retardants (overview), in: J.C. Salomone (Ed.), Polymeric Materials Encyclopedia, vol. 4, CRC Press, Boca Raton, London, 1996, pp. 2397–2411. [20] F.I. Abd-Allah, H.M. Dawaba, M.S.A. Ahmed, Drug Discoveries and Therapeutics 4 (4) (2010) 267–275. [21] K.P. Keshavarao, D. Mudit, D. Lavanya, L.N.V. Krishne, R. Prudhvi, International Research Journal of Pharmacy 2 (11) (2011) 119–123. [22] I.B. Pathan, C.M. Setty, V.R.M. Gupta, Acta Pharmaceutica Sciencia 50 (2008) 219–222. [23] S.S. Ahmad, M. Sabareesh, P.R. Khan, P.S. Krishna, B. Sudheer, Journal of Applied Pharmaceutical Science 1 (8) (2011) 181–185. [24] S.H. Klanga, C.S. Siganosb, S. Benitaa, J.F. Peryb, Journal of Controlled Release 57 (1999) 19–27.
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Optimization and formulation design of carbopol loaded Piroxicam gel using novel penetration enhancers Hema Chaudhary ∗ , Ajay Rohilla, Permender Rathee, Vikash Kumar P.D.M. College of Pharmacy, Sector: 3A, Sarai Aurangabad, Bahadurgarh, Haryana, India
a r t i c l e
i n f o
Article history: Received 6 December 2012 Received in revised form 8 January 2013 Accepted 14 January 2013 Available online 31 January 2013 Keywords: Formulation Drug transport Drug design Box–Behnken design Drug delivery
a b s t r a c t The aim of the study was to develop and optimize Piroxicam transdermal gel formulation using threefactor, three-level Box–Behnken design by deriving a second-order polynomial equation to construct contour plots for prediction of responses as three selected independent variables with ratio of carbopol 974 (X1 ), ratio of propylene glycol (PG) (X2 ) and ratio of ethanol (X3 ). The dependent variables studied were the skin permeation rate of piroxicam (Y1 ), viscosity of the gel (Y2 ) and pH of the gel (Y3 ). Response surface plots were drawn, statistical validity of the polynomials was established to find the compositions of optimized formulation which was evaluated using the vertical Franz-type diffusion cell. The permeation rate of piroxicam increased proportionally with ethanol concentration but decreased with polymer concentration. The design demonstrated the role of the derived polynomial equation and contour plots in predicting the values of dependent variables for the preparation and optimization of gel formulation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Today about 74% of drugs are taken orally and are found not to be as effective as desired. Discovering a new medicine is a very expensive and time consuming process that makes re-designing the modules a more lucrative task. The design of dosage form, whether a tablet, an injection or a patch is to deliver the right amount of medicine at right time to the right target site and it becomes complicated if each medication were to be delivered in an optimal and preferred manner to the individual patient. The medication may not be absorbed if it is released too slowly or if it is delivered too rapidly, the patient may suffer untoward effects and its desired effects may not last as long as needed. Management of illness through various conventional systems of medications involves various problems and complications such as attainment and maintenance of drug concentration in the body within a therapeutic range. It is often necessary to take it several times a day so as to prevent the concentration dropping below the minimum effective level and to exhibit best therapeutic effects. A continuous intravenous infusion is considered as a superior mode of drug administration as compared to oral route. However, to overcome the problems associated with intravenous infusion and to duplicate closely its benefits, skin is used as a most favorable part of drug administration. So, whilst skin is used as a part of drug administration, it is foretold as transdermal administration
∗ Corresponding author. Tel.: +91 8901451603. E-mail address: [email protected] (H. Chaudhary). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.01.015
and the drug delivery through transdermal administration is called as transdermal drug delivery system (TDDS). TDDS is a promising method of drug administration that can avoid the variability in rates of absorption and metabolism encountered in oral treatment [1,2]. Drug Delivery via. the transdermal route is an interesting option because transdermal route is convenient and safe. The positive features of delivering drugs across the skin is to achieve systemic effects by avoidance of first pass metabolism and gastro intestinal incompatibility as it helps to predict and extend duration of activity. TDDS is also used to minimizing undesirable side effects and provides utilization of drugs with short biological half lives with narrow therapeutic window by improving physiological and pharmacological response. This system avoids the fluctuation in drug levels with maintenance of plasma concentration of potent drugs and provides suitability for self-administration and enhanced therapeutic efficacy [3]. There are three approaches for permeation enhancement; drug vehicle based enhancement; physical and chemical enhancement. Chemical enhancement is affected by penetration enhancers [4–7]. They are used for improving transdermal drug delivery by reversibly reducing the barrier resistance. Their mechanisms include disruption of intercellular lipid and/or keratin domains and tight junctions which results in enhanced drug partitioning into tissue or by altering thermodynamic activity/solubility of drug. There are numerous other chemicals that are able to exert the above mentioned effects such as azone, urea, alcohols, surfactants, sulphoxides, glycols and fatty acids. It has been proposed that any chemical that is used in facilitation of transdermal drug delivery should ideally be non-toxic and non-irritating to skin. It should
H. Chaudhary et al. / International Journal of Biological Macromolecules 55 (2013) 246–253
have solubility factor similar to skin and when removed the barrier properties of the skin should reform fully and rapidly. It should be cosmetically acceptable, compatible with wide range of excipients and pharmacologically and chemically inert with no pharmacological action in the body. The terpenes are usually the constituents of volatile oil and categorized in GRAS category by USFDA having pharmacologically inert, non-toxic and non-irritating effect to the skin [8–14]. Terpenes are secondary metabolites that are mostly synthesized in plants. They are found in essential oils consisting of five carbon isoprene unit which undergoes a condensation reaction to forms a much larger (10–40 carbon) terpene frame work. This larger terpene structure can be subjected to further biochemical processing, which produces final range of terpenes. Thousands of different terpenes (monoterepenes, sesquiterpenes and diterpenes) have been identified till date and many have been investigated as penetration enhancers due to their high lipophilicity. They have good safety profile and are non-toxic and non-irritant to the skin. They also possess attributes such as low melting point and low molecular weight. They have high percutaneous enhancement ability, reversible effect on the lipids of stratum corneum, minimal percutaneous irritancy at low concentrations (1–5%). The mechanism of action of terpenes involves increasing one or more of the following effects: diffusion coefficient, partition coefficient, drug solubility (increasing the thermodynamic activity of the drug), lipid extraction (i.e. disruption of lipid–protein domain), macroscopic barrier perturbation and molecular orientation of terpenes molecule within the lipid bilayer. However, the rate and extent of enhancement are dependent upon type and physicochemical characteristics (such as melting point, solubility) of terpene, concentration of terpene used, absence or presence of co solvent or enhancer and concentration of co-solvent or enhancer if present. In recent years, many attempts have been made to investigate the use of terpenes as skin permeation enhancers including menthol, linalool, limonene and carvacrol to promote the transdermal transport of drugs including chiral agents. Monoterpenes were selected due to their small sizes which promote its good penetration enhancer capability. The objective of the study was to develop a low dose transdermal delivery system i.e. transdermal carbopol loaded gels for piroxicam that would provide the release of drug at a controlled rate to achieve a therapeutic drug level for a prolonged period by using myrcene and 3-Carene (monoterpene hydrocarbon are selected since they are lipophilic and smaller in size and a novel approach) as chemical penetration enhancers for the efficacious transdermal delivery of piroxicam.
2. Materials and methods Piroxicam pure was purchased from Taj Pharmaceuticals Limited (India). Carbopol (CP-974) was obtained from Central drug house (Delhi, India). Penetration enhancers (3-Carene and myrcene) were purchased from Sigma Aldrich Chemicals Pvt. Ltd. (UK). Ethanol was purchased from Jiangsu Huaxi International (China). Propylene glycol (PG), methyl and ethyl paraben sodium and sodium hydroxide (pellets) were bought from Central drug house (Delhi, India). All the materials used in the study were of analytical grade and double distilled water was used throughout the study.
2.1. Preliminary trials The experimental field should neither be too large nor too small, leading to nonrealistic experiments from an optimal region [15].
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2.1.1. Selection of polymer For the preparation of gel various polymers were used such as HPMC, carbopol, carboxymethyl cellulose, sodium carboxymethyl cellulose, carboxymethyl mungbean starch, chitosan, etc. with different grades. The carbopol on the basis of its good consistency was used. Many dummy gels were prepared with this polymer at different concentration and the concentrations between 1% and 2% gave good consistency for the gel and it is generally recognized as safe (GRAS) property. Thus, carbopol 974 in the concentration range of 1–2% (w/w) was selected for gel preparation. 2.1.2. Optimization of enhancer Monoterpenes such as myrcene and 3-Carene were selected due to their small size which promotes its good penetration enhancer capability. The working concentration range of the both the enhancer was determined by ex vivo permeation study. The flux was determined from Fick’s law of diffusion considering the transport of drugs across the skin barrier as a process of passive diffusion. The cumulative drug release was calculated as concentration in total volume divided by skin surface area. The flux (g/cm2 /h) was calculated from the slope of the linear portion of the cumulative amount permeated per unit area versus time plot. After ex vivo skin permeation study of piroxicam with different concentrations of both the enhancers, the enhancer was optimized on the basis of cumulative amount of drug release and flux. The steady state permeability coefficient (Kp) and enhancement ratio (ER) were calculated by using following equation: Kp =
J C
where J is the flux and C is the initial concentration of piroxicam. ER = flux of piroxicam containing chemical penetration enhancer/flux of piroxicam without using enhancer. 2.2. Preparation of gels Gels were prepared by dispersing the polymer (Carbopol 974) and slowly added into known quantity of water under constant stirring with addition of methyl paraben sodium (0.01%, w/v) and propyl paraben sodium (0.1%, w/v) and kept for a period of 24 h for soaking. The dispersion was then allowed to hydrate and swell for 60 min. After hydration, drug (0.5%, w/w) was dissolved in appropriate quantity of propylene glycol, ethanol and 12.5% of optimized penetration enhancer i.e. 3-Carene with constant stirring and blend was transferred to container containing soaked carbopol and agitated for additional 20 min. Tri-ethanol amine (TEA) was added in the dispersion and stirred thoroughly until clear viscous homogeneous gel was obtained and pH was kept in the range of 6.0–7.0 for maximum efficiency. The formulations were prepared incorporating CP-974 (1–2%, w/w), PG (5–15%, w/w), ethanol (10–30%, w/w), penetration enhancer (12.5%, v/w) and water using Box–Behnken experimental design and optimized formulation was generated using statistical screening. 2.3. pH evaluation A defined amount of Piroxicam gel (100 mg) was measured and diluted with distilled water and mixed well. The pH of the gel was recorded using Digital pH Meter (Multitech Instrument Co. Pvt. Ltd., Delhi, India) by bringing it in contact with the gel and allowing it to equilibrate for one min. pH evaluation was carried out for all experimental formulations. Experiments were performed in triplicate to check for the neutralization of gels of different batches.
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H. Chaudhary et al. / International Journal of Biological Macromolecules 55 (2013) 246–253 Table 1 Variables with coded values in statistical design.
2.4. Rheological measurements The viscosity measurement of gel was measured by Brookfield viscometer DV-I at 10 rpm (DVS-I Prime, India). Samples of the gels were allowed to settle over 30 min at 25 ± 1 ◦ C before measurements were taken. All viscosity measurements were performed in triplicate. 2.5. Spreadability Spreadability was determined on the basis of slip and drag character of gels after 72 h of preparation. The gel were filled in collapsible tubes and sealed by crimping one of the ends. The weights of the tubes were recorded. The tubes were placed between two glass slides and were clamped. Certain load of weight based on quantity of gel was placed over the slides and then cap was removed. The amount of extruded gel was collected and weighed. The percentage of gel collected was calculated [16]. Spreadability is expressed in terms of time in seconds to slip off from gel and placed in between slides under the direction of certain load. Lesser the time taken for separation of two slides, better the spreadability. It is calculated by using the formula: S=
M×L T
where S represents the spreadability (g/s), M is the weight in pan (g), L is the fixed distance moved by the slide and T is the time. 2.6. Experimental design The formulations were optimization using 3-factor, 3-level factorial design by constructing second order polynomial models and exploring quadratic response surfaces with Design Expert® (version 8.0.7.1, Stat-Ease Inc., Minneapolis, Minnesota). The selected independent and dependent variable with coded levels, low, medium and high levels are listed in Table 1. The experimental design generated polynomial equation, is given as Y0 = b0 + b1 X1 + b2 X2 + b3 X3 + b12 X1 X2 + b13 X1 X3 + b23 X2 X3 + b11 X12 + b22 X22 + b33 X32 where Y0 is the dependent variable; b0 is the intercept; b1 to b33 are the regression coefficients computed from the observed experimental values of Y; and X1 , X2 and X3 are the coded levels of independent variables. The terms X1 , X2 and Xi2 (i = 1, 2, or 3)
Coded levels
Independent variables in % w/w
Low (−1) Medium (0) High (+1)
X1 1 1.5 2
X2 5 10 15
X3 10 20 30
Dependent variables
Constraints
Y1 = flux of piroxicam (g/cm2 /h) Y2 = viscosity of gel (cP) Y3 = pH of gels
32 ≤ Y1 ≥ 110 1182 ≤ Y3 ≥ 3956 6.09 ≤ Y1 ≥ 6.7
X1 = polymer conc. (%); X2 = PG conc. (%); X3 = ethanol conc. (%).
represent the interaction and quadratic terms, respectively [17]. The amount of polymer concentration (X1 ), PG (X2 ) and ethanol (X3 ) used to prepare each of the formulations and their observed and predicted responses are given in Table 2. 2.7. In vitro drug release studies 2.7.1. Preparation of rat skin The animals used for preparation of skin were male albino rats weighing 110–125 g (excised from the abdominal region). The rats were sacrificed by giving excess anesthesia (ether). On the day of experiment the skin was thawed to room temperature and hair and fat were removed. The hair on the abdominal region was carefully removed using hair removal cream and hair removal spatula and adhering subcutaneous fat was cleaned using isopropyl alcohol (IPA) by rubbing with cotton previously dipped in IPA and immediately kept in isotonic phosphate buffer (IPB) solution. This step caused the layer to remain unwrinkled, and then the skin was washed with distilled water [18]. 2.7.2. Permeation studies Ex vivo permeation studies were carried out through rat skin using an automated transdermal Franz vertical diffusion cell sampling system (Rama Glasswares Co. Pvt. Ltd., Delhi, India). The skin was trimmed to the appropriate size and mounted on the diffusion cell such that the stratum corneum of the skin faced the donor cell and dermis faced the receiver cell. The cell was mounted using spring. The receptor compartment was filled with the vehicle (i.e. PEG 400:IPB pH 7.4 (30:70) and the donor compartment was filled with 5 mg drug suspension (0.5%, w/v) containing different concentration of chemical penetration enhancer. The whole assembly was
Table 2 Design of experiments with results. Runs
Batches
X1
X2
X3
Y1 Y2 (Experimental)
Y3
Y1 (Predicted)
Y2
Y3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
GF1 GF2 GF3 GF4 GF5 GF6 GF7 GF8 GF9 GF10 GF11 GF12 GF13 GF14 GF15 GF16 GF17
0 −1 −1 −1 +1 0 0 −1 0 0 0 +1 +1 +1 0 0 0
+1 0 −1 0 0 +1 0 +1 −1 −1 0 0 −1 +1 0 0 0
−1 −1 0 +1 +1 +1 0 0 −1 +1 0 −1 0 0 0 0 0
68.2 99.9 110.5 85.8 21.2 36.1 72.3 102.2 40.8 48.7 75.2 31.9 29.5 52.7 75.1 74.2 76.2
6.29 6.16 6.31 6.09 6.50 6.30 6.30 6.10 6.50 6.50 6.30 6.51 6.70 6.50 6.20 6.30 6.20
68.3 100.0 110.7 85.8 21.0 36.1 72.6 102 40.7 48.5 75.2 32.0 30.2 52.7 75.1 74.2 76.2
3017.1 2338.7 2821.3 2927.2 4248.0 3847.9 3446.1 2756.5 3117.2 3955.9 3488.6 1182.0 3951.9 3808.0 3454.2 3466.1 3459.8
6.30 6.20 6.30 6.09 6.55 6.29 6.20 6.15 6.46 6.47 6.30 6.51 6.70 6.49 6.20 6.30 6.20
3016.5 2338.5 2821.1 2926.8 4248.5 3848.1 3445.7 2756.1 3117.0 3956.1 3489.0 1182.4 3951.3 3807.7 3453.7 3465.7 3460.0
X1 = polymer conc.(%); X2 = PG conc. (%); X3 = ethanol conc.(%); Y1 = flux of Piroxicam gel (g/cm2 /h); viscosity of gel (cP); pH of gels.
All batches of formulated gel were characterized for their cosmetic persona (color, scent, texture and consistency). The formulated gels were light yellowish in color with a pleasurable, smooth homogeneous appearance and texture. The pH value of all the prepared formulations ranged from 6.09 to 6.7 as shown in Table 2. The spreadability plays a considerable role in patient fulfillment and helps in uniform application of gel to the skin. A good gel takes less time to spread and will have high spreadability. The spreadability of formulated gels was decreased as the concentration of polymer increased. The values of spreadability
59.3 73.7 120.4 205.2 307.7 530.4 614.8 778.5 955.5 1208.3 1427.1 1721.4 74.2 0.015 4.22 1.90
GF17 GF16 GF15
60.7 74.7 121.5 206.1 310.3 530.8 619.0 787.4 957.3 1221.9 1429.5 1729.2 75.1 0.015 4.16 1.80 60.5 73.1 146.2 182.2 250.5 409.7 462.3 581.6 630.4 832.8 1048.7 1261.5 52.7 0.011 5.10 2.74
GF14 GF13
0.80 3.84 15.8 40.1 63.2 106.3 150.7 207.9 260.3 396.6 543.2 693.5 29.5 0.006 1.83 3.56 3.03 6.47 19.8 35.2 39.8 62.6 105.3 172.5 223.9 405.3 542.7 721.3 31.9 0.006 3.31 3.50
GF12 GF11
61.9 76.7 123.5 209.5 313.8 536.0 622.3 792.3 957.9 1226.8 1432.4 1734.1 75.2 0.015 4.16 1.73 117.6 184.2 217.4 285.2 329.6 503.2 639.1 719.0 821.4 992.5 1102.4 1209.9 48.7 0.010 3.12 2.91
GF10 GF8 GF7 GF6 GF5 GF4 GF3 Formulation code
3.2. Characterization of gels
Cumulative amount of drug release (g/cm2 )
The ex vivo skin permeation study of piroxicam was performed with different concentration of enhancers. Myrcene at different concentrations such as 5%, 10%, 15% (w/w) did not give good satisfactory results as shown in Table 3. Further, 3-Carene at concentration of 5%, 10%, 15% (w/w) showed that the flux increased in increasing order of enhancer concentration and decreased at 15% (w/w) concentration. The reason of decreased release of drug was due to attainment of saturation point. Thus, the concentration of 12.5% of 3-Carene was selected for further study due to the improved flux (89.26 g/cm2 /h). The satisfactory flux was also not obtained when the mixture of myrcene and 3-Carene were tried which may be due to immiscibility of these enhancers.
Time (h)
3.1. Optimization of enhancers
Table 4 Ex vivo skin permeation study of drug-carbopol loaded gel with optimized penetration enhancer.
3. Result and discussion
GF9
The statistical polynomial equation generated by Design Expert® (version 8.0.7.1, Stat-Ease Inc., Minneapolis, Minnesota) was established on the basis of ANOVA terms in the software. The totals of 17 runs (GF1–GF17) with triplicate center points were generated. The models were evaluated in terms of statistically significant coefficients and R2 values. The compositions of optimized formulation over the whole experimental region were found by conducted validation of RSM results.
4.85 11.3 22.9 36.2 49.6 88.3 168.4 263.6 466.8 708.5 829.5 1054.7 40.8 0.008 3.24 3.10
2.8. Response analysis for optimization
33.2 102.8 145.5 283.6 423.5 573.9 810.3 924.1 1119.8 1500.1 1952.3 3102.5 102.2 0.020 5.36 1.20
placed on magnetic stirrer and stirred magnetically using magnetic bead at 600 rpm placed in receiver cell. Drug permeation studies were carried out for 24 h at a temperature of 37 ± 2 ◦ C. The aliquots of 0.5 ml were withdrawn from the receptor compartment at predetermined time intervals of 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 h and were replaced by the same volume of PEG 400:IPB pH 7.4 (30:70). Receiver volume was immediately replenished with the same volume of fresh vehicle. Permeation parameters like flux (g/cm2 /h) and enhancement ratio were calculated.
58.3 69.0 117.2 200.8 302.9 529.8 607.9 765.0 905.1 1189.9 1350.2 1707.9 72.3 0.014 4.23 2.12
– 2.28 2.36 2.45 2.85 4.47 4.55 4.08 1.33
141.7 189.7 201.6 244.5 279.1 329.9 428.7 466.6 544.1 640.5 724.9 798.4 36.1 0.007 2.88 3.40
0.0039 0.0089 0.0092 0.0095 0.0111 0.0174 0.0178 0.0159 0.0052
22.3 39.5 56.3 66.8 82.8 137.2 147.8 180.2 205.3 317.8 450.4 541.3 21.2 0.004 1.82 3.72
19.59 44.73 46.37 48.09 56.03 87.67 89.26 79.98 26.17
15.2 37.7 83.0 110.9 183.2 288.5 373.9 651.2 724.5 1162 1554.7 1925.1 85.8 0.017 4.81 1.40
Piroxicam (control) Piroxicam + myrcene (5%) Piroxicam + myrcene (10%) Piroxicam + myrcene (15%) Piroxicam + 3-Carene (5%) Piroxicam + 3-Carene (10%) Piroxicam + 3-Carene (12.5%) Piroxicam + 3-Carene (15%) Piroxicam + myrcene (5%) + 3-Carene (5%)
100.8 157.7 297.6 368.2 443.5 630.8 882.2 1076.5 1302.8 1495.8 1821.8 3387 110.5 0.022 5.39 1.00
Enhancement ratio
92.7 115.9 150.7 284.1 355.3 526.9 767.3 841.7 947.3 1265.6 1714.5 1995.5 99.9 0.019 5.16 1.50
Permeability coefficient (Kp)
GF2
Flux (g/cm2 /h)
GF1
Formulation
0.5 33.2 53.0 1.0 2.0 72.1 3.0 125.5 4.0 139.1 6.0 258.7 356.7 8.0 422.9 10.0 12.0 671.3 16.0 890.5 20.0 1105.4 24.0 1508.5 Flux (g/cm2 /h) 68.2 Permeability 0.014 Coefficient enhancement ratio 5.03 2.40 Retained concentration (g/ml)
Table 3 Ex vivo study of piroxicam with chemical penetration enhancer.
64.8 80.2 125.7 220.8 319.1 547.8 636.0 810.9 984.2 1248.5 1438.5 1746.8 76.2 0.015 4.10 1.64
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Table 5 Summary of results of quadratic model for regression analysis of responses Y1 , Y2 , and Y3 . Quadratic model
R2
Adjusted R2
Predicted R2
SD
%CV
Response (Y1 ) Response (Y2 ) Response (Y3 )
0.9992 0.9999 0.9689
0.9982 0.9998 0.9289
0.9980 0.9998 0.8964
1.51 12.62 0.040
1.78 0.04 0.70
[25.83–33.85 (g cm)/s] indicate that the gels were easily spreadable by small amount of shear and possessed acceptable bioadhesion. 3.3. Rheological measurements The viscosity of all the 17 formulations ranged between 1182 and 3951 cP as shown in Table 2. 3.4. Evaluation of in vitro release The effect of optimized penetration enhancer on permeation of piroxicam was evaluated for cumulative amount of drug release. The result of skin permeation studies of drug loaded gel is shown in Table 4. It was found that among all the formulations, GF3 showed highest cumulative amount of drug release (3387.04 g/cm2 ), flux (110.53 g/cm2 /h), permeability coefficient (0.0221) and there is 5.39 folds increase in flux as compared to control gel which is due to reduced inter particular cross linking between drug and polymer causing increase in rate of drug release. The permeation of drug was increasing by using of optimized penetration enhancer i.e. 3Carene as it has potential to solubilize the lipids in stratum corneum without damaging the skin and this leads to enhancement of drug permeation. 3.4.1. Histopathology study A comparative study of penetration enhancing capacity of an optimized formulation gel (GF3) was performed and further compared with the untreated rat skin as shown in Fig. 1. The rat skin section treated with an optimized formulation gel (GF3) showed a significant increase of disruption of stratum corneum organization. The rat skin treated with an optimized formulation gel (GF3) in Fig. 1b showing enlarged intercellular space along with clear reversible disruption of stratum corneum organization i.e. lipid domains, which leads decrease in degree of lipid arrangement in the stratum corneum confirming the penetration enhancement of an optimized formulation. 3.5. Model fitting to the data The total 17 runs with triplicate center points were generated with the help of three factors, three-level Box–Behnken statistical experimental design and responses are observed. The ranges of Y1 is 1–2%, Y2 is 5–10%, and Y3 is 10–30% for all batches and responses flux of gel, viscosity of gel and pH of gel were found to be in the range 32–110 g/cm2 /h, 1182–3956 cP and 6.09–6.7 respectively. The best-fitted model was quadratic and the comparative values of R2 , SD, and %CV are given in Table 5 along with the regression equation generated for each response. All statistically significant (p < 0.05) coefficients are included in the equations. A positive value shows an effect that favors the optimization, while a negative value shows an opposite relationship between the factor and the response. The three independent variables, namely the concentration of CP-974 (X1 ), PG (X2 ), and ethanol (X3 ) has interactive effects on the three responses (Y1 , Y2 and Y3 ).
Fig. 1. High power photomicrograph of section of rat skin (a) untreated; (b) treated with an optimized formulation (GF3).
3.6. Two dimensional plots and response analysis by polynomial equations Two dimensional plots (contour plots) were prepared for all the three responses as shown in Figs. 2–4 for responses Y1 , Y2 and Y3 , respectively. These plots showed the interaction effects of the factors which are useful in studying the effects of two factors on each response at a particular point. 3.6.1. Effect on flux: response 1 (Y1 ) The following polynomial equation was generated for flux of piroxicam Y1 = 74.61 − 32.89X1 + 3.72X2 − 6.10X3 + 7.89X1 X2 + 0.83X1 X3 − 9.99X2 X3 + 5.19(X1 )2 − 6.06(X2 )2 − 20.12(X3 )2 where Y1 is the flux of piroxicam, X1 , X2 and X3 are the concentration of polymer, PG and ethanol respectively. The model F-value of 981.26 implies the model is significant. The lack of fit F-value 95.60 implies the lack of fit is not significant (p ≥ 0.0001). In this case X1 , X2 , X3 , X1 X2 , X2 X3 , X12 , X22 , X32 had a more pronounced effect on flux of piroxicam. The predicted R2 of 0.9980 is in reasonable agreement with the adjustable R2 of 0.9982. The ratio of 101.175 indicates an adequate signal. This model can be used to navigate the design space. The contour plots which show the effect of different independent variables on flux of piroxicam (Y1 ). The amount of polymer increasing will decrease the size of the channels, as does an increase in swelling degree decreases the
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Fig. 2. Contour plot showing the effect of polymer concentration (a) (X1 ) and PG (X2 ); (b) PG (X2 ) and ethanol (X3 ) on response Y1 .
permeation of drug [19]. In our study, it is revealed that flux of piroxicam decreased with increase in concentration of the polymer (CP-974) as the drug remains entrapped in the gel complex of the polymer. The amount of ethanol was not increased beyond 20% as it yielded a hazy product. Flux was found to decrease on increasing the concentration of CP-974 due to increase in viscosity of the gel. A linear relationship was obtained between % (w/w) of PG and flux due to improved drug diffusion as shown in Fig. 2 and flux increased with increase in concentration of ethanol. 3.6.2. Effect on viscosity of gel: response 2 (Y2 ) The following polynomial equation was proposed by the model for viscosity of gel formulation: Y2 = 3462.83 + 543.42X1 − 52.17X2 + 415.61X3 − 19.64X1 X2 + 1119.47X1 X3 − 1.85X2 X3 − 719.59(X1 )2 + 590.81(X2 )2 − 569.17(X3 )2 where Y2 is the viscosity of the gel formulations, X1 is the polymer concentration, X2 is the concentration of PG, and X3 is the concentration of ethanol. The independent factors, polymer concentration is observed to have significantly higher positive effect on the viscosity of the gel due to greater inter particular cross linking between drug and polymer. An inverse relationship was obtained between PG and viscosity due to more spreadability attributed by PG. The viscosity also decreased with increase in concentration of ethanol due to conformational changes in the polymer making the gel. Overall, the model is significant (F-value = 9529.58; p > 0.0001) while the lack of fit is not significant (F-value of 0.08; p = 0.9661). The values for predicted (0.9998) and adjusted (0.9998) R2 values are in reasonable agreement. The signal to noise ratio was found to be satisfactory as the observed adequate precision ratio of 343.618 is above 4. The effect of two independent factors on viscosity at a time as shown in the contour plots (Fig. 3). 3.6.3. Effect on pH of gel: response 3 (Y3 ) The response Y3 relating to pH of gel is shown as: Y3 = 6.26 + 0.19X1 − 0.096X2 − 8.75X3 + 0.015X1 X3 − 2.50X2 X3 + 0.039(X1 )2 + 0.10(X2 )2 + 0.016(X3 )2
where Y2 is the viscosity of the gel formulations, X1 is the polymer concentration, X2 is the concentration of PG, and X3 is the concentration of ethanol. The positive effect of ethanol is found to be more than that of PG on pH. PG does not having any effect on pH of gel but the pH increase with increase in concentration of ethanol as shown Fig. 4. The model F-value of 24.22 implies the model is significant (p > 0.0002) but the lack of fit is not significant. The predicted R2 of response Y3 is 0.8964 which is reasonable agreement with the R2 of 0.9689. The observed adequate ratio was found to be 17.405 is above 4. 3.7. Optimization and data analysis On the basis of physical evaluation and ex vivo skin permeation studies of all control and sample Piroxicam gel formulations, it was found that formulation GF3, GF4 and GF8 gave good results and among these GF3 gave best result. Therefore GF3 was selected as optimized formulation. The optimized formulation of Piroxicam gel (GF3) was compared with marketed formulation for ex vivo skin permeation study as shown in Table 6. The optimized formulation (GF3) showed maximum cumulative amount drug Table 6 Comparative ex vivo skin permeation study of optimized formulation GF3 and marketed formulation of Piroxicam gel. Time (h)
0.5 1 2 3 4 6 8 10 12 16 20 24 Flux (g/cm2 /h) Permeability coefficient (Kp) Enhancement ratio
Cumulative amount of drug permeated (g/cm2 ) GF3
Marketed
100.8 157.7 297.6 368.2 443.5 630.8 882.2 1076.5 1302.8 1495.7 1821.9 3387.0 110.5 0.022 1.67
50.20 60.52 77.73 131.6 144.5 267.8 373.7 433.4 678.7 914.6 1123.1 1490.7 66.10 0.013
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Fig. 3. Contour plots showing the effect of polymer concentration (a) (X1 ) and PG (X2 ); (b) (X1 ) and ethanol (X3 ); (c) PG (X2 ) and ethanol (X3 ) on response Y2 .
release (3387.04 g/cm2 ; flux 110.5 g/cm2 /h) in comparison to marketed formulation (1490.68 g/cm2 ; flux 66.10 g/cm2 /h) with an enhancement ratio of 1.67. 4. Stability studies The accelerated stability analysis for optimized formulation was conducted according to International Conference on Harmonization (ICH) guidelines [20–24]. The optimized gel formulation was stored in well closed glass containers for a period of 90 days at 40 ◦ C temperature and 75% relative humidity in humidity chamber. At predetermined intervals; 0, 30, 60 and 90 days, samples were collected and their physical evaluation parameters such as color, presence of clogs, consistency, phase separation and chemical evaluation parameters such as pH and drug content were evaluated. 5. Conclusion
Fig. 4. Contour plots showing the effect of PG (X1 ) and ethanol (X3 ) on response Y3 .
The Piroxicam gel showed promising results as using carbopol release controlled polymer and optimized using 3-factor, 3-level Box– Behnken design. The quantitative responses; flux of
H. Chaudhary et al. / International Journal of Biological Macromolecules 55 (2013) 246–253
piroxicam, viscosity of gel, and pH of gel for different combinations of independent variables; polymer, PG and ethanol were obtained experimentally and the results were found to fit the design model. The quantitative effect of these factors at different levels on the responses could be predicted by using polynomial. The formulation (GF3), having polymer concentration 1%, PG 5%; ethanol 20%; the flux for formulation was 110.53 (g/cm2 /h) and 2821.15 cP viscosity was chosen as optimized transdermal gel (GF3). Therefore it can be concluded that a transdermal gel formulation for piroxicam was developed and optimized using a 3-factor, 3-level Box–Behnken design. The stability study as per ICH guidelines revealed that the optimized formulation gel (GF3) holds promise for a high stability. Acknowledgements The authors wishes to thank Management of PDM Religious and Educational Association (PDMREA) and also grateful to PDM College of Pharmacy, Sarai Aurangabad, Bahadurgarh, Haryana, India for financial support, and providing the facilities to carry out the research work. References [1] L.V. Allen, N.G. Popovich, H.C. Ansel, Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th ed., Wolter Kluwer Publishers, New Delhi, 2005, pp. 298–299. [2] R. Kumar, A. Philip, Tropical Journal of Pharmacy Research 6 (1) (2007) 633–644. [3] P. Kumar, C. Sankar, B. Mishra, The Indian Pharmacist 5 (3) (2004) 7–17. [4] P.F. Uzor, C.J. Mbah, E.O. Omeje, Journal of Chemical and Pharmaceutical Research 3 (3) (2011) 680–700. [5] B.W. Barry, European Journal of Pharmaceutical Sciences 14 (2001) 101–114.
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