INTRODUCTION:

Penetration enhancers are also known as accelerants, sorption promoter¹ or permeation enhancer². The barrier function is essential for the protective role of stratum corneum but at the same time it may hinder the transdermal delivery of drug through it. As the major route of drug is through the intracellular channels, the lipid section is a viable determinant in the first step of absorption³. Penetration enhancers can temporarily diminish the barrier function of skin to enhance the drug flux. Bisoprol fumarate is an antihypertensive drug with a molecular weight of 767.0 Dalton. The drug idealized for a transdermal patch should be less than 600 Dalton for better permeation through the skin⁴. To cause the penetration of drug with molecular weight greater than 600, penetration enhancers are used which causes disruption of stratum corneum⁵. Thus the present study was conducted to study the effect of permeation enhancers on the drug release profile of Bisoprolol fumarate though excised skin of rabbit at different concentrations. The permeation enhancers selected were non-ionic surfactant (Tween 80), sulfoxide (DMSO) and glycol (Propylene glycol) to understand the effect of enhancement property of different permeation enhancers with different chemical properties.

MATERIAL AND METHOD:

Materials

Bisoprolol fumarate (donated by Mass Pharma, Lahore, Pakistan), Eudragit RS100 (Merck, Germany), Hydroxypropyl methylcellulose E5 (Merck, Germany), Polyethylene glycol 400 (Merck, Germany), Dimethyl sulfoxide (Fisher Scientific), Tween 80 (Daejung, Korea), Propylene glycol 400 (Merck, Pakistan), Polyvinyl alcohol (Merck, Germany), Sodium chloride (Merck, Germany), Potassium dihydrogen phosphate (Fluka, Germany), Disodium hydrogen phosphate (Fluka, Germany), Sodium hydroxide (Riedel-de Haen), Methanol (BDH, England), Hydrochloric acid (BDH, England).

Methods

Preparation of PVA backing layer

A backing layer of 4% Polyvinyl alcohol (PVA) solution was prepared by dissolving PVA in distilled water. Weighed amount of PVA was added in portion in distilled water over 2 hours to ensure complete mixing. Backing solution was poured on the surface of dry petri dish and was allowed to dry completely at room temperature for 24 hours.

Preparation of Bisoprolol fumarate matrix transdermal patch without permeation enhancer (control)

Weighed amount of Hydroxypropyl methylcellulose (HPMC) and Eudragit RS 100 was added in 15ml of methanol followed by addition of Polyethylene glycol 400 (PEG 400) as plasticizer. The solution was stirred on hot plate magnetic stirrer at 32^oC for 60 minutes to ensure complete mixing (Table 1). The drug was dissolved in 5ml of methanol and slowly added to the polymeric solution. The solution was further mixed for 15 minutes for homogenous mixing of drug. The casting solution was poured on the surface of backing layer and a funnel was placed on petri dish in an inverted manner to control the rate of evaporation of solvent⁶. The patches were completely dried at 35°C in oven.

Preparation of Bisoprolol fumarate matrix transdermal patch with permeation enhancer

Weighed amount of Hydroxypropyl methylcellulose and Eudragit RS 100 was added in 15ml of methanol followed by addition of plasticizer and permeation enhancer. The solution was stirred on hot plate magnetic stirrer at 32^oC for 60 minutes to ensure complete mixing (Table 1). The drug was dissolved in 5ml of methanol and slowly added to the polymeric solution. The solution was further mixed for 15 minutes for homogenous mixing of drug. The casting solution was poured on the surface of backing layer and a funnel was placed on petri dish in an inverted manner to control the rate of evaporation of solvent⁶. The patches were dried at 35°C in oven.

Preparation of rabbit skin

The hair on abdominal area of the rabbit was trimmed with an aid of hair clipper. The skin was made hairless by applying hair removal cream for sensitive skin and washed off completely with warm water⁷. The rabbit was sacrificed by cervical dislocation and abdominal region was obtained. The epidermis was prepared by soaking the skin in water at 60°C for 45 seconds⁶. The sub-dermal tissues were removed with forceps and dermis side was wiped for 1 minute with a cotton swab dipped in Isopropyl alcohol (IPA) to remove adhering fats from the surface⁷. The skin was washed with warm distilled water, kept in saline solution and stored in refrigerator. It was used within one week of preparation. Before starting the experiment the skin was allowed to reach room temperature for at least 10 hours⁸ and equilibrated for 1 hour in phosphate buffer saline pH 7.4⁹.

In vitro skin permeation

The in vitro skin permeation study of films across rabbit skin was conducted in Franz diffusion cell. The dermal side of skin was placed facing the receptor compartment. A circular transdermal patch was pressed on the skin with backing layer side facing away from the stratum corneum. The receptor compartment was filled with phosphate buffer saline pH 7.4. The system was connected to a thermostatically controlled water bath to maintain temperature at 32±2 °C by circulating water through a jacket surrounding the cell body⁷. After every one hour a sample of 0.5 ml was withdrawn from the receptor compartment and replaced with an equal volume of phosphate buffer saline pH 7.4. The sample was diluted with appropriate volume of fresh phosphate buffer saline pH 7.4 and analyzed spectrophotometrically at 223 nm. A blank patch without drug was prepared and treated similarly on Franz cell to get blank solution for UV analysis.

Data analysis

Calculations for in vitro skin permeation studies

The in vitro skin permeation studies were analyzed for cumulative amount of drug permeated, flux and permeability coefficient.

Cumulative amount of drug permeated in µg/cm²was plotted against time. Drug flux in µg/cm².hr at steady state was calculated by dividing the slope of linear portion of curve by the area of the exposed skin surface i.e. 1.2 cm². The permeability coefficient in cm/hr was deduced by dividing the flux with initial drug amount¹⁰.

Calculation of enhancement ratio

Permeation enhancement ratio which is also known as enhancement factor or enhancement index was determined by¹¹:

ER= (Drug permeability coefficient after enhancer treatment/ Drug permeability coefficient before enhancer treatment)

Table 1: Formulation of optimized matrix type transdermal patches with permeation enhancer

Formulation code	ERS 100:HPMC	Bisoprolol fumarate (mg)	PEG 400 (40% w/w)	Penetration enhancer	Methanol (ml)
Control	9:1	10	400	-	20
F01	9:1	10	400	Tween 80 (10%)	20
F02	9:1	10	400	Tween 80 (20%)	20
F03	9:1	10	400	Tween 80 (30%)	20
F04	9:1	10	400	Tween 80 (40%)	20
F05	9:1	10	400	PG (10%)	20
F06	9:1	10	400	PG (20%)	20
F07	9:1	10	400	PG (30%)	20
F08	9:1	10	400	PG (40%)	20
F09	9:1	10	400	DMSO (10%)	20
F10	9:1	10	400	DMSO (20%)	20
F11	9:1	10	400	DMSO (30%)	20
F12	9:1	10	400	DMSO (40%)	20

ERS 100: Eudragit RS100

RESULT AND DISCUSSION:

The permeation profile of Bisoprolol fumarate in presence of Tween 80, PG and DMSO is shown Fig. 1-3 respectively. The flux, permeability coefficient and ER of permeation enhancers at different concentrations are tabulated in Table 2.

In case of Tween 80 (Fig. 1), the highest permeation of Bisoprolol fumarate was obtained at 40% concentration. When the concentration of Tween 80 was increased it decreased interfacial tension and increases wetting of polymer to a greater extent thus erosion of polymer occur (Sonjoy et al., 2011). In case of Tween 80 ER was greatest in formulations containing 40% Tween 80. Thus it was evident from the data that as concentration of Tween 80 increased from 10% to 40% the permeation enhancing effect also increased. Tween 80 at 10% concentration failed to enhance the effect as permeability coefficient of control patch was greater than that of F01. This signified that at this concentration Tween 80 has lesser ability to allow the passage of Bisoprolol fumarate through the stratum corneum. Tween 80 is a non-ionic surfactant and contains ethyleneoxide and long chain hydrocarbon chain that imparts both hydrophobic and hydrophilic characteristics. This attribute allows the partitioning between both lipophilic lipid molecules and hydrophilic protein domain. The other mechanism by which Tween 80 is believed to increase the rate of drug release is by penetrating into intracellular matrix followed by interaction and binding with keratin filament which causes disruption of the corneocytes¹². This makes the area more aqueous and changes partition coefficient of the region¹³. It was generally recognized that non-ionic surfactants possesses least toxicity and skin irritation potential as compared to anionic, cationic and zwitterionic surfactants¹.

Fig. 1: Cumultive amount of drug permeated from Bisoprolol fumarate matrix transdermal patch containing Tween 80 as permeation enhancer (n=3)

Table 2 shows that the presence of PG produced the highest permeation rate at concentration of 30% (Fig. 2). Increasing the concentration from 30% to 40% reduces the permeation rate and the flux reduced. In case of PG the greatest ER was at 30% concentration and was better than that observed in Tween 80. When the concentration of PG was increased from 10% to 20% no significant increase in enhancing property was observed (Table 2). However at 30% concentration of PG there was a drastic increase in ER. This is due to the fact that at higher concentration PG acts both as an enhancer and as a cosolvent. As cosolvent it solubilizes the drug and increases diffusion rate¹⁴. When the concentration was further increased by an increment of 10%, the ER was 2.856. This indicated that PG at concentration of 30% is better suited to increase the flux as compared to 40% PG. This result has been verified in the studies of Carvidilol matrix transdermal patch where marked ER was observed at 30% concentration of PG as compared to 25%, 35% and 45%¹⁵. The permeation of PG alters thermodynamic activity of drug in system and modifies driving force for diffusion. It partitions into the tissue facilitating uptake of drug into skin and implements some minor disturbance to intercellular lipid packing within the stratum corneum¹⁶. PG acts as penetration enhancer by solvent drag mechanism i.e. it carries drug into the tissues rather than fluidizing the lipids¹⁷.

Fig. 2: Cumultive amount of drug permeated from Bisoprolol fumarate matrix transdermal patch containing PG as permeation enhancer (n=3)

Table 2: Slope, flux, permeability coefficient and enhancement ratio (ER) of Bisoprolol fumarate matrix patch containing permeation enhancers

Formulation code	Slope	Flux (µg/cm²hr)	Permeability coefficient (cm/hr)	ER
Control	296	247	0.0274	--
F01	157	131	0.0145	0.53
F02	364	304	0.0337	1.23
F03	591	492	0.0547	1.99
F04	753	627	0.0697	2.54
F05	334	279	0.0310	1.13
F06	362	301	0.0335	1.22
F07	911	759	0.0843	3.08
F08	844	703	0.0782	2.86
F09	386	322	0.0358	1.31
F10	714	595	0.0661	2.41
F11	740	617	0.0685	2.50
F12	577	482	0.0536	1.96

In case of DMSO an increase in the concentration of sulfoxide resulted in an increase in the permeation rate of Bisoprolol fumarate (Fig. 3) and highest permeation rate was obtained at 30% DMSO. Table 2 shows that increasing the concentration of DMSO from 10% to 30% caused an increase in ER where as at 40% concentration a reduction in ER was observed. DMSO acts as a permeation enhancer by denaturing intercellular structural proteins of horny layer and promoting lipid fluidity by disruption of lipid chains in the skin. DMSO also alters the physical structure of stratum corneum by extraction of lipids, lipoprotein and nucleoproteins in the skin structure¹⁸.

Fig. 3: Cumultive amount of drug permeated from Bisoprolol fumarate matrix transdermal patch containing DMSO as permeation enhancer (n=3)

Comparing different concentrations of various permeation enhancers, it is clear from Table 2 that the transdermal patch containing 30% PG leads to the highest flux value of Bisoprolol fumarate. The ERs of different concentrations were 0.530-3.079 with PG showing the most potent enhancing effect (759.14 µg/cm²hr), followed by Tween 80 (627.37 µg/cm²hr) and DMSO (616.66 µg/cm²hr) with 30%, 40% and 30% concentration respectively.

The plot of ER versus concentration of the permeation enhancer is shown in Fig. 4. The figure showed that in all cases, except Tween 80, the greatest enhancement of the skin transport occurs at 30% concentration of the enhancer, but this is seen to decrease at higher concentration (40%). The previous studies on Bisoprolol fumarate for drg delivery system have also confirmed the passage of drug through animal membrane using different combination of polymers¹⁹ and plasticizer²⁰. It has been reported that animal membranes tend to be considerably more fragile than human skin membranes. Thus, the action of aprotic solvent (DMSO) on animal tissue may be dramatically greater as compared to the effects seen on a human skin membrane¹⁶. On the other hand, non-ionic surfactant (Tween 80) effect on transdermal flux is comparatively small at lower concentrations¹³.

Fig. 4: The effect of permeation enhancer on the ER of Bisoprolol fumarate through rabbit skin

CONCLUSION:

It could be reasonably concluded from the above data that the concentration of permeation enhancers plays an important role in the ER through the excised rabbit skin. Permeation enhancers can be used at definite concentrations to increase the flux of drugs with higher molecular weight. Human skin membrane can be used to further study the effect of flux of Bisoprol fumarate through the transdermal patch.

REFERENCES:

1. Songkro S. An overview of skin penetration enhancers: penetration enhancing activity, skin irritation potential and mechanism of action. Songklanakarin J. Sci. Technol. 31(3); 2009: 299-321.

2. Karande P and S Mitragotri. Enhancement of transdermal drug delivery via synergistic action of chemicals. Biochimica et Biophysica Acta. 1788(2009: 2362-2373.

3. Rowat AC et al. Interactions of oleic acid and model stratum corneum membranes as seen by ²H NMR. International Journal of Pharmaceutics. 307(2006: 225-231.

4. Hupfeld S and H Gravem. [Transdermal therapeutic systems for drug administration]. Tidsskr Nor Laegeforen. 129(6); 2009: 532-533.

5. Benson HAE. Transdermal drug delivery: penetration enhancement techniques. Current Drug Delivery. 2(2005: 23-33.

6. Limpongsa E and K Umprayn. Preparation and evaluation of Diltizem hydrochloride diffusion-controlled transdermal delivery system. AAPS PharmSciTech. 9(2); 2008: 464-470.

7. Xi H et al. Transdermal patches for site-specific delivery of anastrozole: In vitro and local tissue disposition evaluation. Int J Pharm. 391(1-2); 2010: 73-78.

8. Ren C et al. Design and in vivo evaluation of an indapamide transdermal patch. Int J Pharm. 370(1-2); 2009: 129-135.

9. Prabu SL et al. Design and evaluation of matrix diffusion controlled transdermal patches of Dexibuprofen. J App Res. 12(1); 2012: 38-46.

10. Gannu R et al. Development of nitrendipine transdermal patches: in vitro and ex vivo characterization. Curr Drug Deliv. 4(1); 2007: 69-76.

11. Mutalik S et al. A combined approach of chemical enhancers and sonophoresis for the transdermal delivery of tizanidine hydrochloride. Drug Deliv. 16(2); 2009: 82-91.