A novel aspiration in treatment of chronic disease like diabetes associated with other non communicable disease risk factors, such as hypertension is to provide greater therapeutic effect, overcome the side effects by complex therapeutic regimen and to improve patient compliance upon administering combinational transdermal delivery of Glibenclamide (G) and Atenolol (A) which have not been tested literally. Hence, it is aim to develop a transdermal patch containing Glibenclamide and Atenolol using different polymer combinations such as Hydroxy propyl methyl cellulose (HPMC), Poly vinyl pyrolidone (PVP) and Carbopol (CP). The developed patches were evaluated for physicochemical parameters, in-vitro and in-vivo drug release and in-vitro skin permeation studies. Good results were obtained in all the evaluated parameters. The drug release of all formulation follows zero order kinetics by diffusion mechanism of non fickian diffusion type. In-vitro transdermal permeation studies by using rat & goat skin and finally in-vivo studies by using rabbits were carried out for the optimized formulation (GA4 HPMC 1%, PVP 0.5%, CP 0.5%). The developed Transdermal delivery system containing Glibenclamide & Atenolol might be a milestone in the combinational therapy of diabetes and hypertension.
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Keywords: Transdermal patches, Glibenclamide, Atenolol, diabetes, hypertension.
Many of the initial goals for transdermal drug delivery have been selectively achieved with currently marketed products, such as providing a convenient, painless method of drug delivery, improving patient compliance, reducing adverse delivery, reducing adverse effects and maintaining more consistent and prolonged blood levels than those achieved with oral or parenteral dosing. The technology was quickly accepted by patients and clinicians alike, and patches were viewed as a desirable platform for a variety of therapeutic uses, including motion sickness, hypertension, and angina, hormone therapy, smoking cessation and pain control (Robert 2006).
Diabetes mellitus is a cardiovascular disease according to the American Heart Association (Grundy et al., 1999). The major adverse outcomes of diabetes mellitus are a result of vascular complications, both, at the micro vascular and macro vascular levels (Grundy et al., 1998). Furthermore, these complications are amplified by the co-existence of hypertension (Epstein et al., 1992). So, treatment should not only target lowering of blood glucose level, but should also focus on the correction of other non communicable disease risk factors, such as hypertension.
Patients are often to take multiple medications with these conditions and they fail to stick on to the medical regimen because of the number of medicines required (Kroenke et al., 1991). The more medications given, the lesser patients adhere to the full treatment or course of therapy. If both drugs given in a single formulation the total number of medications taken per day can be reduced.
Glibenclamide (G) is a popular anti-diabetic drug, belonging to the class of sulfonylurea. The drug is widely used for treating type II diabetes. The common side effects after oral therapy are gastric disturbances like nausea, vomiting, anorexia and increased appetite. Patient compliance is very important, beacause these drugs are usually intended to take for a long period (Srinivas mutalik et al., 2005).
Atenolol (A), a Î²-blocker, is prescribed far and wide in diverse cardiovascular diseases, eg, hypertension, arrhythmias, angina pectoris and myocardial infarction. It has been reported that administration of conventional tablets of atenolol exhibit fluctuations in the plasma drug levels results reduction of drug concentration at the receptor site (Bhupinder Singh et al., 2006). Evidence that diabetic patients may experience greater cardio protection with Î²-blockers than do nondiabetic patients (Vivian Fonseca et al., 2008).
In this work it is designed to develop 24 hours transdermal therapeutic system of G and A with the following objectives to overcome gastrointestinal incompatibility and cardiac adverse effects, to avoid hepatic first pass metabolism (Clemett et al., 2000) to simplify treatment regimen, reduce the frequency of administration, overcome the side effects, and to obtain greater therapeutic efficacy to improve patient compliance.
MATERIALS AND METHODS
Glibenclamide obtained from Sri Raghavendra Chemicals and Suppliers, Bangalore. Atenolol obtained from Indian Drugs, Hyderabad. Hydroxy Propyl methyl cellulose (HPMC K4M), PVP (K30), Carbopol (934P) obtained from Indian Drugs, Hyderabad. All other chemicals were of analytical grade.
Determination of partition Coefficient:
The partition co-efficient of the drugs was determined using n - Octanol: Water system which is a parameter of lipophilicity. Atleast 24 h before the experiment, n- Octanol and water were presaturated. In a separating funnel, an accurately weighed quantity of each drug was dissolved in 10 ml of the n-octanol phase against 10 ml aqueous phase and shaken at 37Â°C for 24 h periodically. The separated n-octanol phase was assayed by UV spectrophotometer to determine its residual concentration in the aqueous phase (Marin et al., 1998, McDaid et al., 1996). The partition coefficient was expressed as ratio of concentration of drug in the n-octanol phase (% w/v) to the concentration in the aqueous phase.
Drug-Polymer interaction study
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The IR spectra of the pure drugs, Glibenclamide and Atenolol and a mixture of polymers, HPMC, PVP and, CP were taken in the range of 400-4000 cm-1 using potassium bromide disc method using Thermo Nicolet USA, FTIR instrument.
Fabrication of transdermal patches
Transdermal patches were fabricated using different polymers containing combination of Glibenclamide and Atenolol were prepared by Solvent Casting technique. Base materials were added into the drug solution in ethanol (Yanli Gao et al., 2009, Samanta et al., 2002). Plasticizer and permeation enhancers were added to the solution, and then agitated in a sonicator. This was casted on a glass surface containing ring, it was covered by funnel to control evaporation of solvent and allowed to dry at room temperature over night. The patches were separated and the backing membrane used was aluminium foil and the formulations were stored in desiccator. After being dried, the single-layer patch was obtained. The composition of patches was mentioned in table 1.
Physico chemical evaluation of the prepared patches
Thickness and Weight Variation
The thickness of the patch was determined by using thickness gauge and the patches were then weighed individually using digital balance to determine the weight of each patch taken out from the casted patch. The patches were subjected to weight variation by individually weighing ten randomly selected patches. These was carried out for three times for each formulation. (Mundada et al., 2009).
The patches of specified area were taken into 100 ml phosphate buffer (pH 7.4) in volumetric flask and were sonicated (Mazzo et al., 1994). A blank was prepared in the same manner using a drug-free placebo patch of same dimensions. The solution was then filtered and the drug content was analyzed at 229 nm and 275 nm respectively by UV spectrophotometer.
Folding endurance was carried out by folding the patch at the same point for number of times till it broke (Ubaidulla et al., 2007). Folding endurance ensures the efficiency of the plasticizer and the strength of the patch prepared using varying ratios of the polymers. The test was carried out in triplicate.
Percentage Moisture Loss
Accurately weighed patches of each formulation 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 (Kusum Devi et al., 2003). The test was carried out in triplicate. It was calculated as the difference between initial and final weight with respect to initial weight.
Percentage Moisture Uptake
Accurately weighed patches of each formulation were placed in a desiccator at 79.5% relative humidity (aluminium chloride saturated solution) at room temperature and weighed after 3 days (Biswajit Mukherjee et al., 2005). The test was carried out in triplicate. It was calculated as the difference between final and initial weight with respect to initial weight.
Water Absorption Capacity
Three patches of each formulation were kept in an atmosphere of relative humidity RH = 82% for one week and the difference in the weight of the patches was taken as the water absorption capacity for that patch (Udupa et al., 1992).
Water Vapor Transmission Rate
Transmission cell of equal diameter were used for water vapor transmission studies (Kulkurni Raghavendra et al., 2000). 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 deccator 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
The in-vitro release for patches was carried out by using Chein apparatus. The receptor compartment was maintained at 37 Â± 1Â°C by means of water. The freshly prepared phosphate buffer pH 7.4 placed in the receptor compartment was continuously stirred at 60 rpm by means of Teflon coated magnetic stirrer, in order to avoid effects of diffusion layer. The Commercial Semi-permeable membrane were mounted between the two compartments and held in place by means of a clamp. The combination patch was placed on one side of the semi-permeable membrane (Ji-Hui Zhao et al., 2007, Yanli Gao et al., 2000). Aliquots of 1mL were removed from the receptor compartment and it is replaced immediately with the same volume of buffer solution. Samples were taken from the medium at regular time intervals over a period of 24 hours and the samples were analyzed for Glibenclamide and Atenolol content by UV spectrophotometer at 229 nm and 275 nm respectively (Vlassios Andronis et al., 1995).
In-vitro Transdermal permeation
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The hairs of the male wistar albino rat were cleared by using scissors. After cleaning the skin with Phosphate buffer pH 7.4, animal was sacrificed by excessive ether inhalation. An incision was made on one side of the animal and the skin was separated. The prepared skin was washed with Phosphate buffer pH 7.4 and used (Jain et al., 2003, Yanli Gao et al., 2009).
Skin (approximate 1mm in thickness) was obtained from slaughtered goat. The skin was removed carefully and separated. After separating skin, the fat adhering to the dermis side was removed using isopropyl alcohol.
The transdermal permeation was performed in Chein apparatus. The cells were filled with freshly prepared phosphate buffer pH 7.4. The patch is placed on stratum corneum side of skin and dermis side was facing receptor compartment (Ke et al., 2005). Receptor compartment contains phosphate buffer pH 7.4 and samples were taken at regular time intervals and it is replaced with receptor fluid. The samples were analyzed at 229 nm and 275 nm against blank by UV spectrophotometer (Srinivas Mutalik et al., 2006).
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 (Draize et al., 1944), 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 Study
Selection of animals
Rabbit's (crytolagus cuniculus) of male sex 10-12 weeks old weighing 1-2 kg were selected. They were kept with husk bedding and were fed with standard rodent pellet diet and water. 12 hours light (Light cycle) and 12 hours dark (dark cycle) were maintained. The temperature and relative humidity conditions were 28o C Â± 2 o C and 60 Â± 15% respectively. The protocols for all animal studies were approved by Institutional Ethical Committee (1220/a/08/CPCSEA/ANCP/06).
A set of healthy rabbits were selected. They were checked to ensure that they were free from disease. The hair was removed from the dorsal surface of the selected rabbits. The dose of Glibenclamide and Atenolol was calculated according to the body weight i.e., 0.32mg and 3.2mg respectively (Jayaprakash et al., 2010). The patch GA4 (HPMC 1%, PVP 0.5%, CP 0.5%) was placed on the dorsal surface. At specific time interval the patch was removed from the rabbit carefully and analyzed for remaining drug content. Initial drug content was determined before placing the patch. The remaining drug content was subtracted from the initial drug content of the patch. The value obtained indicates the amount of drug in diffused from the patch into the body (Chakkapan et al., 1994).
Amount of the drug released at any time interval = Initial drug content before placing the patch - Remaining Drug content after removal of the patch
In-vitro In-vivo correlation
In-vitro and In-vivo correlation was carried out for the therapeutic efficacy of a pharmaceutical formulation. It is governed by the factors related to both in-vitro and in-vivo characteristics of the drug. Percent in-vitro release on X-axis was plotted against in-vivo drug release on Y-axis for the same period of time.
RESULTS AND DISCUSSION
In the present work efforts have been made to prepare Combinational transdermal delivery of Glibenclamide and Atenolol using combination of polymers such as Hydroxy Propyl methyl cellulose, Carbopol and Poly vinyl pyrrolidone. Permeation enhancer used was Propylene glycol.
Partition coefficient of Glibenclamide and Atenolol was found to be 3.16 and 2.14 respectively, which is favorable for the transdermal delivery.
The physicochemical compatibility of the drugs and the polymer was established through FTIR studies. In the physical mixture of Glibenclamide and atenolol with Hydroxy propyl methyl cellulose, Carbopol and Poly vinyl pyrrolidone the major peaks of Glibenclamide and Atenolol were 1714, 1638 (C =O Stretching), 1415 (CH2 Bending), 1342, 1300 (SO2 Asymmetric Stretching), 1244, 1158 (C - N Stretching) wave numbers. The spectra indicated that there was no chemical interaction between Glibenclamide and Atenolol and other excipients which are shown in figure 1-3.
Physico-chemical evaluation data of table 2 and 3 indicates that thickness of patches varied from 0.30 Â± 0.01 to 0.38 Â± 0.04. The drug content analysis and the weight uniformity of the prepared formulation have shown that patches in this investigation with uniform drug content and with minimum intra batch variability.
Folding endurance values of matrix patches was found within 230 - 286 numbers of folds, indicating good elasticity and maintain the integrity.
The percentage Moisture uptake in the formulation GA2 (1% HPMC, 1% CP) has shown the highest value of moisture absorption 12.21Â±0.02 which may be due to the presence of high hydrophilicity of HPMC and CP.
The formulation GA5 (0.5% HPMC, 1% PVP, 0.5% CP) shows higher value of Moisture loss 11.26Â±0.032 which is due to presence of higher concentration of PVP and formulation GA2 (1% HPMC, 1% CP) shows low value of 4.12 Â± 0.015.
The high water absorption capacity was found in GA4 (1% HPMC, 0.5% PVP, 0.5% CP) as 12.24Â±0.01 which also revealed its high hydrophilicity. The formulation GA1 (1% HPMC, 1% PVP) has shown maximum water vapor transmission of 9.987 X 10-6 among all the patches this may be due to the presence of more PVP and GA6 has less water vapor transmission due to high amount of Carbopol 3.588 X 10-6.
The in-vitro drug release data in table 4 indicated that formulation GA1 (1 % HPMC, 1% PVP) has shown release 99.1% and 99.58% respectively at 20th hour. The in-vitro drug release plot has shown that the drug release followed zero order kinetics, which was envinced from the regression value of the above mentioned plot. Peppa's plot shown a slope value of 0.608 and 0.650 respectively, which confirms that the diffusion mechanism involved in the drug release was of non - fickian diffusion type.
The formulation GA2 (1% HPMC, 1%CP) and GA3 (1% PVP, 1%CP) has shown release 98.68%, 99.6% and 99.9% and 98.99% respectively at 19th hour. The drug release was diffusion mediated and from the Peppa's plot shown slope value of 0.575, 0.6442 and 0.595, 0.493 respectively, it is confirmed that it is of non-fickian type.
The formulation GA4 (1%HPMC, 0.5% PVP, 0.5%CP) has shown the drug release of 96.24% and 97.21% at 24th hour respectively. The in-vitro drug release plot which is shown in figure 4 indicated that the drug release followed zero order kinetics, which was envinced from the regression value of the above mentioned plot. The Higuchi's plot has shown the regression value of 0.994 and 0.995 respectively, which indicated that diffusion mechanism influencing the drug release. This substantial increase may be due to the slow dissolving nature of HPMC and less amount of PVP and CP than HPMC, which might have facilitated more drug release from the patch. Peppa's plot was drawn which has shown slope value of 0.686 and 0.590 respectively, which indicates the drug release was of non - fickian diffusion type.
The formulation GA5 (0.5%HPMC, 1% PVP, 0.5%CP), has shown the drug release of 92.32% and 93.25% at 24th hour respectively. Peppa's plot shown slope value of 0.710and 0.707 respectively, it is confirmed that it is of non-fickian type. The decrease in drug release GA5 compared to GA1, GA2, GA3 and GA4 is due to the nature of the polymer. Polyvinyl pyrrolidone has better control release of drug when compared with Hydroxy Propyl methyl cellulose and Carbopol.
The formulation GA6 (0.5%HPMC, 0.5% PVP, 1%CP) & GA7 (0.7%HPMC, 0.7% PVP, 0.6%CP), has shown the drug release of 99.38%, 98.1% and 99.34%, 98.42% respectively at 22th hour. The drug release was diffusion mediated and of non - fickian type. The drug release was diffusion mediated and from the Peppa's plot which has shown slope value of 0.641, 0.674 and 0.794, 0.824 respectively, it is confirmed that it is of non-fickian type
The in-vitro release plots of all other formulations were suggestive of zero order release and are diffusion mediated which was envinced form the regression value Higuchi's plot. All the formulations undergo non-fickian type of release which is confirmed form the slope values obtained from the Peppa's plot. Based on the drug release the optimized formulation selected for further study was GA4 (1% HPMC, 0.5% PVP, 0.5% CP).
In-vitro transdermal permeation study was carried out in rat skin, the formulation GA4 (HPMC 1%, PVP 0.5%, CP 0.5%) showed drug diffusion for 24 hours up to the extent of 93.24% and 94.26% respectively. The studies, which were carried out with goat skin showed drug diffusion of and 92.92% and 93.64% respectively and these were mentioned in table 5. The variation among the used biological membrane may be due to the fat content and thickness of the membrane used. As the goat skin has more fat deposition and the thickness compared with rat skin, it might have hampered the drug release through the membrane.
The result obtained from the primary skin irritation studies revealed that neither the adhesive nor the drugs Glibenclamide and Atenolol caused any noticeable irritation on the rabbit skin throughout the study.
In-vivo study was carried out in rabbit revealed that the formulation GA4 in-vitro release was reproducible even in biological environment. At the end of 24th hour the in-vivo drug release showed 90.62 % and 91.25% respectively and values were mentioned in table 5. The results which are mentioned in table 6 indicated that the in-vitro and in-vivo techniques correlation was very good. They are well correlated, so the release pattern has followed the predicted zero order kinetics in biological systems also which are shown in figure 5-6.
In conclusion formulation GA4 (1%HPMC, 0.5% PVP, 0.5%CP) has achieved the targets of present study such as controlled release, prolonged zero order release, reduced frequency of administration, greater therapeutic effect, overcome the side effects, simplify the treatment regimen and thus may improve patient compliance.
The authors are very much thankful to the management of Annamacharya College of pharmacy for affording the feasibilities to carry out the research work and Sri Raghavendra Chemical and Suppliers for providing the gift sample of Glibenclamide.