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The partition coefficient of MX was carried out in n-octanol/phosphate buffer pH 7.4 (9). The two phases were shaken together initially to ensure mutual saturation. An accurately weighed quantity of MX was dissolved in 10ml of the n-octanol phase and shaken at 25Â°C for 24 h against 10 ml aqueous phase in a sealed container. The separated n-octanol phase was assayed by UV spectroscopy to determine its residual concentration and hence the amount partitioned into the aqueous phase. The partition coefficient was expressed as the concentration of drug in the n-octanol phase (% w/v) divided by the concentration in the aqueous phase (10).
MX was determined by High Performance Liquid Chromatography (HPLC) (11). Separation was achieved on a Chromosil LC - 18 (250mm X 4.6mm internal diameter, 5ïm particle size) eluted with a mobile phase consisting of Methanol : Phosphate Buffer pH 6.0 adjusted with Sodium hydroxide (50:50) delivered at a flow rate of 1 ml/min. Detection wavelength was 346 nm. The injection volume was 20 Î¼l. Typical retention time was 4.4 min. Peak area was used to determine MX concentration. Calibration curves were linear in the range 0.1-0.8 mcg/ml.
Drug-Polymer Interaction Study
The Drug and polymer interaction between MX and polymers used in the films were studied by using Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared (FTIR) Spectroscopy.
Fabrication of patches:
Transdermal patches were fabricated using different polymers containing MX by Solvent Casting technique (12, 13). Adhesive patches containing MX were prepared by dissolving polymers individually or in combinations in suitable solvents namely dichloromethane and ethanol. Propylene glycol (30%v/v) of polymer composition was used as a permeation enhancer. The solution was poured into a glass ring which is covered with funnel. The solvent was allowed to evaporate at ambient conditions for 24 h. The patches were then covered with backing membrane cut into appropriate sizes, packed in aluminium foil and stored in a desiccator for further studies. Table 1 enlists the composition of different formulations prepared using varying amounts of the polymers.
Physico Chemical Evaluation of the Prepared Patches
Thickness and Drug content
The thickness of the patch at three different points was determined using thickness gauge and the patches. Films of specified area were cut and weighed accurately (14). Pieces were taken into a 100 ml volumetric flask containing phosphate buffer (pH 7.4), and the flask was sonicated for 8 h (15). And the solution concentration is found in respective nm.
Folding endurance test
Folding endurance test was carried out by folding the patch at the same point a number of times till it broke (16). The test was carried out to check the efficiency of the plasticizer and the strength of the film prepared using varying ratios of the polymers. The test was carried out in triplicate.
Accurately weighed films of each formulation (n = 3) were kept in a desiccator which is maintained at 79.5% relative humidity (saturated solution of aluminium chloride) at room temperature and weighed after 3 days (17). The percentage of moisture uptake was calculated as the difference between final and initial weight with respect to initial weight.
Accurately weighed films of each formulation (n = 3) were kept in a desiccator and exposed to an atmosphere of 98% relative humidity (containing anhydrous calcium chloride) at room temperature and weighed after 3 days (18). The percentage of moisture loss was calculated as the difference between initial and final weight with respect to initial weight.
Tensile strength and Percentage elongation at break
Mechanical properties of the film were evaluated using an "Instron Tensile Strength tester" (Series IX Automated Material Testing System). A film strips with the dimension (15 cm x 7.5 cm) and free from air bubbles (or) physical imperfections was prepared and cut it in a Dumbell shape, before fed into the equipment. This test was carried out with 50% humidity at 20oC (19). The crosshead speed employed were 100mm/min, with the full-scale load range of 500 Kgf. The force and percentage elongation were measured, when the films were broken. Measurements were run in three replicates for each formulation.
Water Vapor Transmission Rate
Transmission cell of equal diameter were used for water vapor transmission studies (20). These cells were thoroughly washed and dried in an oven. About 1 gm of calcium chloride anhydrous was placed in cell and the patch was fixed over the rim with the aid of the solvent. They were accurately weighed and placed in a desiccator containing potassium chloride saturated solution to maintain 84% RH humidity. The cells placed in desiccator were removed and weighed after 1, 2, 3, 4, 5, 6 and 7th day.
W V T = WL/S
Where, W is transmitted water vapor in mg, L is patch thickness in mm, S is exposed surface area in cm2.
In-vitro Drug Release Studies:
A Franz diffusion cell was used for drug release study from the formulations (21, 22). Phosphate buffered saline (PBS; 20 ml, pH 7.4) was used as the receptor fluid. The receptor fluid was agitated at 600 rpm by a Teflon-coated magnetic stirrer. The semi permeable membrane was mounted between the donor and receptor compartment. The MX drug matrix was placed on one side of the dialysis membrane. The receptor compartment was surrounded by a water jacket to maintain a temperature at 37 Â± 0.5Â°C during the drug release study. Samples were collected from the sampling port at every six hour and were replaced with equal volume of fresh receptor fluid. The collected samples was subjected HPLC to determine the content of the MX.
In-vitro Skin Permeation Studies
Ethical clearance for the handling of experimental animals was obtained from the Institutional animal ethical committee (IAEC) formed for this purpose. The approval number was 661/05/c/CPCSEA AWD dt 07.06.2005/KMCP/IIECA/018/08 dt 19.01.2008.
Snakeskin (23) offers considerable advantages over human material, as it is relatively abundant. In shed snake, the permeability co-efficient of lipophilic drugs was in the same range as those through the human skin. Shed skin of "NAJA NAJA" was collected and soaked in pH 7.4 Phosphate buffer for half an hour and then used. The shed skin was mounted in such a way that the ventral surface side of the skin was kept intimate contact with the formulation and keeping the dorsal region of skin being contact with the release surface of the donor compartment.
Albino porcine ear (24) was obtained from a local slaughter house. The epidermis was prepared by a heat separation technique. The whole skin was soaked in water at 60°C for 45 seconds, followed by careful removal of the epidermis. The epidermis was washed with pH 7.4 Phosphate buffer and used.
The in-vitro skin permeation studies were also carried out using Franz diffusion cell. The temperature of receptor phase was maintained at 37 Â± 1 Â°C throughout the experiment. The compartment was in contact with the ambient environment. The amount of drug permeated through the above mentioned various animal skins were determined by withdrawing samples of 1 ml at predetermined time intervals. The same volume of freshly degassed buffer was supplemented to the receptor after each sampling. The samples were filtered through a 0.2Âµ filter membrane, and an aliquot of 20 Âµl filtrate containing MX was assayed by HPLC method.
In-Vivo Studies on Rabbits
Primary Skin Irritation Test:
The dorsal part of rabbit was shaved carefully, and patch was applied on that skin for 7 days. Conditions of the skin were observed after the patch was removed and are evaluated most often by modification described by Draize (25), which is based on scoring method. Scores as assigned from 0 to 4 based on the severity of erythema or oedema formation. The safety of the patch decreases with increase in scoring.
In-vivo drug release:
Protocols for all animal experiments were approved by Institutional Animal Ethics Committee or Biosafety committee, ANCP. Four Male Rabbit's (Corytolagus cuniculus) of 10-12 weeks old weighing 2-3kg were selected (26). They were kept with husk beeding and were fed with standard rodent pellet diet and water. Light and dark cycle with 12 hrs light and 12hrs dark was maintained. The temperature, relative humidity conditions were 28oC ï‚± 2oC and 60ï‚±15% respectively. The dorsal surface of the selected rabbits was cleaned and hair were carefully shaven using a hair clip per and an electric razor. The dose of MX for oral medication was 1mg/kg. The animal dose of MX was calculated according to the body weight as 0.528 mg. The area each patch sample was 2 cm Ã- 2 cm. Patch samples were applied on the shaved site in the dorsal surface. Patch samples were applied on the shaved site in the dorsal surface. At specific intervals of time, the films were removed carefully and analyzed for the remaining drug content, by HPLC method. The drug content obtained was subtracted from the initial content in the film. The value obtained denotes the amount of drug released in the rabbits (27).
Drug at any interval time = Initial drug content - Final drug content
(Before placing film) (After removal of the film)
Carrageenan induced edema model
The ant-inflammatory activities were found out against Carrageenan induced paw edema in rats. Male albino rats weighing approximately 200-250 g were divided into 3 groups and each group consists of 6 animals (28). One day before the experiment, the left hind thigh of each animal was shaved without damaging the skin. The patch samples were applied to the shaved area in the left hind thigh. The first group (control) received orally 0.5ml of normal saline solution. The second (standard) group received Diclofenac sodium patches. The third groups received prepared MX transdermal formulation respectively. The dose was calculated as 0.133 mg. 60 minute before patch application 0.1 ml of 1% Carrageenan in isotonic saline was injected subplantarly into left hind paw. The volume of the left hind paw was measured using a displacement of plethysmometer.
Stability studies were performed for 3 months using MX prepare patches (29,30). Prepared patches (formulation M2) were kept in refrigerator, stability chamber and incubator for maintaining the temperature of 40C, 400C and 600C respectively. At specific interval of time 15, 30, 45, 60, 75, 90th day the patches were allowed to determine the drug content by HPLC method.
Monolithic drug-in-adhesive transdermal drug delivery system containing MX was prepared by using different polymeric ratios of hydroxyl propyl methyl cellulose, poly vinyl pyrrolidone and hydrophobic polymer of ethyl cellulose in combination or individual.
The thermograms of pure drug and physical mixture of drug with excipient are presented in figures 1and 2. FTIR techniques have been used here to study the interactions between drug and excipients used. The Infra Red (IR) spectra of MX, HPMC, EC, PVP and physical mixture of MX patch formulation are presented in figures 3 - 7.
Table 2 and 3 lists various physico-chemical parameters computed for all the formulations. The thickness of the patches varied from 0.29 Â± 0.01 to 0.35 Â± 0.04. Folding endurance values of matrix films were found within 255 - 282 no of folds, indicating good strength and elasticity and can maintain the integrity with general skin folding. The percentage moisture uptake in the formulation MX - 3 (1% EC) has shown the lowest value of moisture absorption 1.614 Â± 0.032. The formulation MX - 2 (2% HPMC) shows higher value of moisture loss 6.143 Â± 0.01. Formulation MX - 3 (1% EC) shows low value of 1.858 Â± 0.01.The formulations MX - 4 (2% EC) have shown lowest value of 275.55 & 0.295 for tensile strength and percentage elongation respectively. The formulation MX - 2 (2% HPMC) has shown maximum water vapor transmission of 9.297 X 10-6 among all the patches.
Table 4 lists the cumulative percentage drug release of various formulations. The cumulative percentage of drug released in 24 h was found to be the highest (99.28%) from formulation MX - 2. Figure 8 exhibits the dissolution profile obtained for formulation MX - 2. The Higuchi's plot has shown the regression value of 0.998, which indicated that diffusion mechanism influencing the drug release. In order to confirm this fact, Peppa's plot was drawn which has shown slope value of 0.690, which confirms that the diffusion mechanism involved in the drug release was of non - fickian diffusion type. Hence formulation MX - 2 was selected as the optimized formulation by virtue of its drug release kinetics.
The in-vitro skin permeation of MX -2 was performed using various biological membranes such as Snake shed skin and porcine ear skin showed drug diffusion of 24 h up to the extent of 95.52% and 88.20% respectively. Table 4 lists the in-vitro skin permeation of various biological membranes. Further, it was also showed no significant difference among the different barriers used. Figure 9 exhibits the in-vitro skin permeation of MX -2. Between barriers used the Snake shed skin was found to be more permeable.
The safety of MX-patch was evaluated with primary skin irritation study. The results indicated that neither the adhesive nor the drug MX caused any noticeable irritation on the rabbit skin throughout the study. In-vivo study was carried out in rabbit revealed that the consistence in-vitro release pattern of the formulation MX - 2 was reproducible even in biological environment. Table 4 lists the In-vivo drug release of formulation MX - 2. At the end of 24th hour the in-vivo drug release showed 98.19 % release.
The correlation between the results obtained by the in vitro and in vivo techniques was very good. To ensure the correlation between the in-vitro and in-vivo release pattern, the regression analysis was carried out. Figure 10 exhibits the correlation between the in-vitro and in-vivo release pattern.
Foot edema induced by carrageen was effectively suppressed by MX patch. The result obtained in this study showed that the percentage paw edema inhibition was 47.38% for animal treated with MX patch and 48.04% for animal treated with Diclofenac sodium patch. Table 5 lists the Anti Inflammatory Activity data for formulation MX - 2.
The formulation (MX - 2) was subjected to accelerated stability testing at 40C, 400C and 600C for 90 days. The formulation was found to be stable with respect to MX assay when analyzed by HPLC. Period of expiry was found to be 186 days at 250C could be assigned to the TDDS.
Transdermal delivery offers several advantages over oral routes for controlled drug delivery viz., ability of drug for a longer time than the oral dosage forms, evading of hepatic first-pass metabolism, avoid the chemical or metabolic degradation, the delivery of the API can be immediately discontinued (e.g., upon occurrence of adverse reactions).
Meloxicom (molecular weight: 351.4) showed favorable partition coefficients and negligible skin degradation. Hence in this study, the monolithic patches of Meloxicom containing different concentrations of hydroxyl propyl methyl cellulose, poly vinyl pyrrolidone and hydrophobic polymer ethyl cellulose in combination or individual.
The possible drug-polymer interaction was studied by IR and DSC of placebo films and MX loaded matrix films. The results indicated that drug and polymer were compatible for the development of transdermal drug delivery system.
The thickness and drug content analysis of the prepared formulation have shown that the process adopted for casting the films in this investigation is capable of giving films with uniform drug content and with minimum intra batch variability. Folding endurance values of all formulations indicates good strength and elasticity and can maintain the integrity with general skin folding.
The percentage moisture uptake in the formulation MX - 3 (1% EC) has shown the lowest value of moisture absorption which may be due to the hydrophobic nature of the polymer used in it. The formulation MX - 2 (2% HPMC) shows higher value of moisture loss which is due to its hydrophilic nature and formulation MX - 3 (1% EC) shows low value which is due to its hydrophobic nature. The formulations MX - 4 (2% EC) have shown lowest values of tensile strength and percentage elongation respectively, when compared with other formulations and it is clearly found that the tensile strength and the percentage elongation increase as the polymer content in the patch decrease. The formulation MX - 2 (2% HPMC) has shown maximum water vapor transmission. This may be due to the presence of high hydrophilic nature of the polymer.
In vitro release test is widely used because of its simplicity and reproducibility. Moreover, it has been showed that drug diffusion through matrix was influenced by the drug-polymer interaction. However, in vitro tests are very useful in the quality control of finished TDDS. Drug released in 24 h was found to be the highest for formulation MX - 2 and diffusion mechanism involved in the drug release was of non - fickian diffusion type. Hence formulation MX - 2 was selected as the optimized formulation by virtue of its drug release kinetics.
The in-vitro skin permeation of MX -2 was performed using various biological membranes such as Snake shed skin and porcine ear skin which showed drug diffusion up to 24 h. Further, it was also showed no significant difference among the different barriers used. The drug diffusion of MX from Snake shed skin at all the concentrations tested was found to be more in comparison to porcine ear skin. As the Porcine ear has more fat deposition and thickness, it might have hampered the drug release through the membrane.
Since patch is applied on skin, the local safety, skin irritation should be confirmed. The results indicated non-irritating level, thus, MX-patch is expected to be applied on skin without skin irritation. In-vivo study was carried out in rabbit revealed that the consistence in-vitro release pattern of the formulation MX - 2 was reproducible even in biological environment. The sustained response following transdermal administration was due to controlled and continuous release of drug into the systemic circulation over an extended period. They are well correlated, so the release pattern has followed the predicted zero order kinetics in biological systems also.
Foot edema induced by carrageen was effectively suppressed by MX patch. The result obtained in this study showed that the percentage paw edema inhibition was almost similar for animal treated with MX patch and for animal treated with Diclofenac sodium patch.
Less degradation and good physical appearance was observed on performing the stability studies and period of expiry was found to be 186 days at 250C could be assigned to the TDDS.
The transdermal formulation and the prototype patch were shown to be efficacious, safe, stable and non-irritant to skin. The formulation MX - 2 (2% HPMC) has shown optimum release in concentration independent manner. While release kinetics (Higuchi's plot) of drug is the first-order process, suggesting that the outwards moving of drug from the adhesive is associated with the diffusion process. Good correlation is observed between in-vitro and in vitro skin permeation profile, which reveals the ability of the formulation to reproduce the in-vitro release pattern through various biological membranes. In-vivo studies, carried out using rabbit affirmed that the reliability of the in-vitro evaluation methods followed and the adaptability of the delivery system to the biological environment for MX - 2. Pharmacodynamic studies revealed that the anti-inflammatory activity of the Transdermal formulation MX - 2 shows appreciable results when compared with standard Diclofenac patch. The Shelf life of the formulation MX - 2 was found to be 186 days. Further studies, will deal with the application of the presently reported findings to human skin permeation, involving in vivo tests. The obtained results are encouraging for the further development of this novel drug delivering technology for the skin.
The author wishes to thank the management of K. M. College of pharmacy for carrying out the skin permeation studies and Annamacharya College of Pharmacy for providing all facilities to carry out the remaining part of the research work. The author is grateful to Sun pharmaceuticals ltd, India for the gift samples of Meloxicam.