Design , Formulation and Evaluation of Transdermal Drug Delivery System of Budesonide

Budesonide is a highly potent synthetic, nonhalogenated corticosteroid. The mechanism of action of corticosteroids in allergic rhinitis remains unknown, but may involve reductions in number of various mediator cells such as basophils, eosinophils, T-helper cells, mast cells, and neutrophils. In the nasal mucosa, nasal reactivity to allergens, and release of inflammatory mediators and proteolytic enzymes. Budesonide is very effective and quikly acting as it is rapidly and almost completely absorbed after oral administration, but has poor systemic availability (about 10%) due to extensive first-pass metabolism in the liver, mainly by the cytochrome P450 isoenzyme CYP3A4. The major metabolites, 6-βhydroxybudesonide and 16-α-hydroxyprednisolone have less than 1% of the glucocorticoid activity of unchanged drug with a terminal half-life of about 2 4 hours. Polymeric films containing Eudragit RL 100: Eudragit RS: drug (7:3:1, 7: 2:1) and Ethyl cellulose: PVP: drug (7:3:1, 7:2:1) were selected for transdermal administration based on evaluation studies. These polymeric films were prepared by mercury substrate method employing PEG-400 as plasticizer. Two different penetration enhancers Urea and Dimethyl sulphoxide (DMSO) were employed in the study. The patches in each group were uniform in drug content, thickness. In Vitro drug permeation, moisture absorption and WVTR studies were carried out on these test patches. It was found that at all humidity condition the absorption increases which were linear to the moisture absorbed. In PVA and EUDRAGIT RL 100 patches the water vapor transmission rate was found to be higher at 75% RH, RT conditions. Therefore at both % RH, RT condition the PVA and EUDRAGIT RL 100 patches provide the best resistance to water vapor. Therefore, when applied to animals (in further studies) these patches may provide more occlusion to water vapor loss from skin thus making atmosphere beneath the skin more humid that aid in drug permeation.


Introduction
Corticosteroids and their biologically active synthetic derivatives differ in their metabolic (glucocorticoid) and electrolyte-regulating (mineralocorticoid) activities.These agents are employed at physiological doses for replacement therapy when endogenous production is impaired.In addition, glucocorticoids potently suppress inflammation, and their use in a variety of inflammatory, asthmatic conditions, skin disorders, rhinitis, inflammatory bowel disease, collagenous colitis and autoimmune diseases makes them among the most frequently prescribed classes of drugs [1].Budesonide is a highly potent synthetic, nonhalogenated corticosteroid.It has high glucocorticoid and weak mineralocorticoid activity [2].Exact mechanism(s) of action of corticosteroids in allergic rhinitis remains unknown, but may involve reductions in the following: number of mediator cells (basophils, eosinophils, T-helper cells, mast cells, and neutrophils) in the nasal mucosa, nasal reactivity to allergens, and release of inflammatory mediators and proteolytic enzymes [3].
Budesonide is rapidly and almost completely absorbed after oral administration, but has poor systemic availability (about 10%) due to extensive first-pass metabolism in the liver, mainly by the cytochrome P450 isoenzyme CYP3A4 [4].The major metabolites, 6-β-hydroxybudesonide and 16-α-hydroxyprednisolone have less than 1% of the glucocorticoid activity of unchanged drug with a terminal half-life of about 2 -4 hours [4][5][6].
The polymeric film containing Eudragit RL 100: Eudragit RS: drug (7:3:1, 7:2:1) and Ethyl cellulose: PVP: drug (7:3:1, 7:2:1) were selected for transdermal administration based on evaluation studies [7,8].The polymeric films were prepared by mercury substrate method employing PEG-400 as plasticizer.Two different penetration enhancers Urea and Dimethyl sulphoxide (DMSO) were employed in the study.The dried polymeric film was evaluated using different parameters including thickness uniformity, drug content of the film, in vitro drug release from films and in vitro skin permeation of drug, prior to their in vivo evaluation [8,9].

Animals
The Swiss albino rats (170 -190 gm) were obtained from National Chemical Labortary, Pune, India, and maintained at 25 ± 1˚C for the study.The animals were housed in stainless metabolic cages and provided with standard diet and water ad libitum.Necessary approvals were obtained from CPCSEA India, for conducting the study.

Preparation of Transdermal Patch by Solvent
Casting on Mercury Substrate [10,11] The transdermal patch was prepared by solvent evaporation technique on mercury substrate.Polymer solution was prepared in ethanol (10 ml) and to it budesonide was added.The plasticizer or permeation enhancer or the pore forming agent were added during patch casting.The solution was poured on glass rings placed on mercury surface and allowed to dry in air for 24 hours.Circular patches of 2 cm diameter (3.14 cm 2 ) were cut from semidried patches and placed in desiccator with 0% Relative Humidity (RH).

Evaluation of patch
1) Measurement of thickness [11] Thickness was measured using micrometer screw gauge.Each patch was measured for thickness at five different points to ascertain thickness uniformity in patch.
2) Drug content [11] Accurately weighed patches were individually dissolved in minimum quantity of ethanol and volume was made up to 20 ml with pH 7.4 phosphate buffer containing 2.5% ethanol.From this solution, 1 ml was transferred to volumetric flask and volume was made up to 10 ml.The absorbance was recorded at 247 nm.The blank solution was prepared in the similar way except that the patches without drug were used.
3) Moisture absorption studies [12] The moisture absorption study was carried out at 43, 75, 93% RH, RT at 25 ± 1 °C.The pre-weighed samples of patches were kept under the humidity conditions and weighed after 24 hours.The increase in weight indicates the moisture absorption by samples.
4) Dissolution studies [13,14] The accurately weighed patches were fixed on glass discs of 2.5 cm diameter using standard glue.This assembly was kept in dissolution vessel such that the patch faced the dissolution media upwards.The dissolution media was 900 ml of pH 7.4 phosphate buffer containing 2.5% ethanol at 32 ± 0.5˚C.The speed of paddle was kept at 50 rpm.Samples were withdrawn at 1 hour interval till 12 hours and replaced with the media.The absorbance was measured at 247 nm against the blank.5) In vitro skin permeation studies [10] Cellophane membrane was used.The membrane was mounted between the donor and receptor compartment of Franz diffusion cell.The patch was kept in contact with cellophane membrane.The receptor compartment contained pH 7.4 phosphate buffer containing 2.5% ethanol.The assembly was kept on a magnetic stirrer and stirred at a speed of 200 rpm.The temperature of assembly was kept at 37 ± 1˚C.After each hour, 1ml of sample was withdrawn and replaced with same media up to 24 hours to study.
6) Effect of drugs loading and polymer concentration on film [15] Films with different drug load and polymer concentration were prepared and studied for their thickness, moisture absorption, and In vitro drug permeation.
7) Effect of penetration enhancers on film Two penetration enhancers namely urea, dimethylsulphoxide (DMSO) were incorporated in different proportion 5 and 10%.The films were then evaluated for the properties as mentioned earlier.
8) Effect of plasticizers on film Two plasticizers namely polyethylene glycol-400, glyceryl triacetate were added in different concentration 5 and 10 % of polymer concentration.These films were then evaluated for the properties as mentioned earlier.
where, Qt-amount of drug released at time t; Qo-initial amount of drug.and Ko, K1, KH, KHC and KK are the coefficients of equations.9) Water vapor transmission rate (WVTR) The WVTR study was carried out in desiccators main-tained at 43 and 75% RH at 25 ± 1˚C using saturated solution of potassium carbonate and sodium chloride respectively.Patches were placed on the mouth of glass vials containing fused calcium chloride and sealed using silicon wax.These vials were accurately weighed and placed in desiccators at 0% RH.The weight of these vi-als was recorded after 24 hours.The increase in weight was indicative of water transmission across the patch.
10) Study of drug release kinetics [16] In order to investigate the drug release mechanism from patches, the % cumulative drug release data was analyzed with following mathematical model.
The most appropriate model was selected on the basis of goodness of fit test.The zero order kinetic describes the systems in which the drug release rate is independent of its concentration.The drug releases slowly (assuming that the area does not change and no equilibrium conditions are obtained).The first order kinetics describes the systems in which drug release rate is concentration dependent.Higuchi model describes the release of watersoluble drug from an insoluble matrix as a diffusion process based on the Fick's law and is square root time dependent.
The Hixson-Crowell cube root law describes the drug release from a system depends upon the change in surface area or diameter of particle or system and involves no diffusion mechanism.Korsmeyer-Peppas model describes the fraction of drug release relates exponentially with respect to time.This model is generally used to analyze the release of pharmaceutical polymeric dosage forms, when the release mechanism is not well known or when more than one type of release phenomena could be involved.
11) Skin irritancy study [17,18] Area on the back of rats was clean shaved 24 hours prior to testing.Optimize patch was applied to the clean area and kept in its place by adhesive tape.After every hour, the patch was removed and observed visually for signs of edema or erythema and scored according to Draize's scoring index.Patch without drug was used as control patch.

Table 1. Effect of Drug Loading and Polymer Concentration on Film
The patches in each group are uniform in drug distribution.The thickness and weight increases with the increase in polymer concentration.The films formed are transparent in appearance.(Tables 1 and 2) 3.2.day, however the patches were found to be less stable at 93% RH, RT on 5th day.

Table 4. Moisture Absorption of Eudragit RL
100 Patches For Eudragit RL100 patches at 43% RH, RT the Patches lost their shape on 7th day, while at 75% RH, RT the Patches lost their shape on 5th day and at 93% RH, RT patch were still more unstable and lost their shape at 4th day.
Since the absorption pattern is not uniform in both the cases therefore no conclusion can be drawn regarding the stability of patch from the above data.At every % RH, RT condition the Eudragit RL 100 patches absorb less moisture than PVA patches, but PVA patches were found to be more stable than the Eudragit RL 100 patches.

In Vitro Drug Permeation through Cellophane Membrane
The in-vitro permeation of PVA patches was studied by using cellophane membrane, results obtained are as shown under;  The in vitro drug permeation in all the cases increase up to 7% w/w polymer concentration while at 8% it decreases with constant drug load, the permeation of drug increases with increase in polymer content up to 7% w/w and thereafter it decrease.With constant polymer concentration, higher drug load gives higher permeation of drug.Thus the maximum percent permeation in V, F,  and J series was observed with 7% w/w polymer concentration and it was 15.05%, 14.86%, 14.32% respectively as represented by Figure 1, 2 and 3 for PVA concentration.

In Vitro Drug Permeation through Cellophane Membrane
The drug permeation through cellophane membrane was studied, results obtained are as shown under;     The in vitro drug permeation in all the cases increase upto 6% w/w polymer concentration & there after it decreases.Higher drug load gives higher permeation of drug.thus the maximum percent permeation in EU2, EL2, EB2 series was observed with 6% w/w polymer  concentration and it was 15.05%, 14.86%, 14.32% respectively after 24 hrs.PVA 7% w/w gives maximum drug permeation while EUDRAGIT RL 100 at 6% w/w gives maximum permeation of drug.The maximum % permeation with PVA was 15.05 while with EUDRAGIT RL 100 it was 15.44%.For same drug content, the permeation through EUDRAGIT RL100 was better and higher than with PVA.Also with constant polymer concentration the drug permeation by EUDRAGIT RL100 was higher than PVA.Therefore, with EUDRAGIT RL 100 better permeation may be obtained with less polymer requirement in in-vitro studies, therefore, EUDRAGIT RL 100 was better polymer than PVA.

Effect of Penetration Enhancer on Film
The compositions of various films and patches obtained were examined and characterized for the parameter are shown as below;

Table 11. Compositions of Patches with Different
Penetration Enhancers.

Table 12. Characterization of PVA & EUDRAGIT RL 100 with Different Penetration Enhancers
In both the cases the patches formed are uniform with respect to drug content also with increase in the amount of penetration enhancer the weight of the patch also increase linearly .The thickness was also to be uniform throughout the patch.J3.The moisture absorption increases with increase in enhancer content.DMSO 10% w/w gives greater absorption than other i.e. 22.39%.It is found that 75% RH, RT the absorption pattern is higher than at 43% RH, RT.At 75% RH, RT maximum absorption is shown by urea 10% w/w with 49.47% absorption & at 95% RH, RT with 59.36%.All humidity condition the absorption increases with increase in enhancer content.As the humidity increases, there increase in moisture absorption and this increase linear.However, at 43% RH, RT all the patches retain their shape at the end of 7th day, At 75% RH, RT the urea patches lose their shape on 7th day, while DMSO patches a little sticky to touch.At 95% RH, RT the urea patches lose their shape on 4th day while DMSO patches still sticky & lose their shape on 6th day.
It is found that around 43% RH, RT, the moisture absorption by EUDRAGIT RL 100 patches is comparable with patch without the enhancers i.e.EB3 urea 10% w/w absorb moisture more than any other.When the conditions were of 75% RH, RT and at 95% RH, RT the absorption pattern was found to be higher than at 43% RH, RT At 75% RH, RT maximum absorption is shown by urea 10% w/w with 19.42% absorption & at 95% RH, RT with 24.015.Around all the humidity condition the absorption increases with increase in enhancer content.As the humidity increases, there increase in moisture absorption and this increase linear.
At 43% RH, RT all the patches retain their shape at the end of 7th day, At 75% RH, RT the urea patches lose their shape on 6th day, while DMSO patches a little sticky to touch.At 95% RH, RT the urea patches lose their shape on 3rd day while DMSO patches still sticky & lose their shape on 6th day.
Therefore amongst the enhancers, the patches of UREA & DMSO are stable for 7 days under different humidity condition.

In Vitro Drug Permeation through Cellophane Membrane
The patches were then subjected to in vitro drug permeation through cellophane membrane and the results obtained are indicated as below; The result obtained is compared with patches without        penetration enhancers used in different proportions urea at 10% w/w concentration gives maximum drug permeation i.e. of 27.38% with the enhancement ratio of 2.01.Therefore for budesonide in EUDRAGIT RL 1OO matrix urea 10% w/w is the best penetration enhancer.For both PVA & EUDRAGIT RL 100 patches urea is the best penetration enhancers than DMSO.

Effect of Plasticizers on Films
The compositions of various films are shown as follows; 3.9.1.Table 18.Compositions of Patches with Different Plasticizers The patches obtained were examined and characterized for the parameter as shown below

Table 19. Characterization of PVA & Eudragit
RL 100 Patches with Different Plasticizers In both the cases the patches formed are uniform with respect to drug content also with increase in the amount of plasticizers the weight of the patch also increase linearly.The thickness was also to be uniform throughout the patch.

Table 20. Moisture Absorption Study of PVA
and EUDRAGIT RL 100 Patches with Different Plasticizers At 43% RH, RT, the moisture absorption by PVA patches is comparable with patch without the enhancers i.e.J3.The moisture absorption increases with increase in plasticizer content.PEG-400 10% w/w gives greater absorption than other i.e. 59.87%.Around 75% RH, RT the absorption pattern is higher than at 43% RH, RT.At 75% RH, RT maximum absorption is shown by PEG-400 10% w/w with 50.75% absorption & at 95% RH, RT with 59.87%.In all humidity condition the absorption increases with increase in plasticizer content.As the hu- As the humidity increases, there increase in moisture absorption and this increase linear.At 43% RH, RT all the PEG-400 patches retain their shape at the end of 7th day, while glyceryl triacetate patches lost their shape on 6th day.At 75% RH, RT the PEG-400 patches lose their shape on 6th day, while glyceryl triacetate patches lost their shape on 5th day.At 95% RH, RT the PEG-400 patches lose their shape on 4th day while glyceryl triacetate patches still sticky & lose their shape on 3rd day.5% & 10% w/w of PEG-400, the permeation increased from 20.94% to 24.33% .Similarly with glyceryl triacetate the permeation increased from 19.42% to 22.54% resp.thus the PEG-400 with 10% gives maximum permeation hence it is selected for final formulation.At all humidity condition the absorption increases.As At all humidity condition the absorption increases.As the humidity increases, there is a increase in moisture absorption and this increases linearly.The patch FP-1 gives maximum absorption at 93% RH, RT i.e. 62.46 than any other.At 43% RH, RT the PVA patches retain their shape at the end of 7th day, At 75% RH, RT the PVA patches lose their shape on 7th day, At 95% RH, RT the PVA patches lose their shape on 5th day.patches lost their shape at the end of 7th day, At 75% RH, RT the EUDRAGIT RL 100 patches lose their shape on 6th day, At 95% RH, RT the EUDRAGIT RL 100 patches lose their shape on 4th day.In PVA and EUDRAGIT RL 100 patches the water vapor transmission rate was found to be higher at 75% RH, RT conditions.Therefore at both % RH, RT condition the PVA and EUDRAGIT RL 100 patches provides the best resistance to water vapor.Therefore, when applied to animals (in further studies) these patches may provide more occlusion to water vapor loss from skin thus making atmosphere beneath the skin more humid that aid in drug permeation.

Figure 1 . 3 . 4 . 5 .Figure 2 .
Figure 1.Effect of PVA concentration with constant drug concentration (15 mg) on drug permeation.3.4.5. Figure 2. Effect of PVA Concentration with Constant Drug Concentration (10 mg) on Drug Permeation 3.4.6.Figure 3.Effect of PVA Concentration with Constant Drug Concentration (5 mg) on Drug PermeationThe in vitro drug permeation in all the cases increase up to 7% w/w polymer concentration while at 8% it decreases with constant drug load, the permeation of drug increases with increase in polymer content up to 7% w/w and thereafter it decrease.With constant polymer concentration, higher drug load gives higher permeation of drug.Thus the maximum percent permeation in V, F,

Figure 2 .
Figure 2. Effect of PVA concentration with constant drug concentration (10 mg) on drug permeation.

Figure 3 .
Figure 3.Effect of PVA concentration with constant drug concentration (5 mg) on drug permeation.

Figure 7 .
Figure 7. Effect on drug permeation from PVA patches 5 (Mg) with different urea concentration.

Figure 11 .
Figure 11.Effect of different Conc. of PEG-400 on drug permeation from PVA matrix.

Figure 12 .
Figure 12.Effect of different Conc. of Glyceryl Triacetate on drug permeation from PVA matrix.

Figure 13 .
Figure 13.Effect of different Conc. of PEG-400 on drug permeation from EUDRAGIT RL 100 matrix.

Figure 14 .
Figure 14.Effect of different Conc. of GLYCERYL TRI-ACETATE on drug permeation from EUDRAGIT RL 100 Matrix.

1 .
Preparation and Evaluation of Patch Using Optimum ConcentrationComposition of various film prepared is shown in

. Table 13. Moisture Absorption by PVA and EUDRAGIT RL 100 Patches with Different Penetration Enhancers
At 43% RH, RT, the moisture absorption by PVA patches is comparable with patch without the enhancers i.e.

Table 15 . Drug Permeation from PVA Patches 5 (Mg) with Different DMSO Concentration Percent Permeated Time (Hrs) Enhancement CODE
concentration gives maximum drug permeation i.e. of 25.92% with the enhancement ratio of 1.810.Therefore for budesonide in PVA matrix urea 10% w/w is the best

. Figure 14. Effect of Different Conc. of Glyceryl Triacetate on Drug Permeation from EUDRAGIT RL 100 Matrix The
results are compared with patches without plasticizers i.e.J3.The PVA patches with 5% & 10% w/w of PEG-400, the permeation increased from 16.50% to 19.96%.Similarly with glyceryl triacetate the permeation increased from 17.56% to 23.76 % respectively thus the glyceryl triacetate with 10% gives maximum permeation hence it is selected for final formulation.The results are compared with patches without plasticizers i.e.EB3.The EUDRAGIT RL 100 patches with

Table 23 .
3.12.1.1.Table23.Composition of Films of PVA and EUDRAGIT RL 100 The patch obtained was studied and their characterization was done and is listed in following

Table 24 .
3.12.1.2.Table24.Characterization of PVA and EUDRAGIT RL 100 Patch The patch in each group is uniform in drug content and thickness through the patch.And further In Vitro drug permeation, moisture absorption and WVTR studies were carried out.