INTRODUCTION:

Transdermal delivery of drugs through the skin to the systemic circulation provides a convenient route of administration for a variety of clinical indications. Pharmaceutical scientists have accepted the challenge of transdermal drug delivery over the last 25 years. Most recently, there is an increasing recognition that the skin can also serve as the port of administration for systemically active drugs. In this case, the drug applied topically will be absorbed first into blood circulation and then be transported to target tissues. This could be rather remote from the site of drug application, to achieve its therapeutic purpose ^[1]. Recently, it is becoming evident that the benefits of i.v. drug infusion can be closely duplicated, without its hazards, by using the skin as the port of drug administration to provide continuous transdermal drug infusion into the systemic circulation.

The oral route of administration has certain disadvantages such as destruction of drugs by hepatic first pass metabolism and enzymatic degradation within the gastrointestinal tract. Continuous intravenous administration at a programmed rate has been recognized as a superior mode of drug delivery not only to bypass hepatic first pass effect, but also to maintain a constant, prolonged and therapeutically effective drug level in the body ^[2].

The simply designed transdermal patch has undergone a dramatic transformation over the past decade. All transdermal systems attempt to create a balance between a number of key factors including size of patch or coverage area, concentration of the drug, duration of therapeutic drug level and use of a skin penetration enhancer ^[3].

The transdermal drug delivery systems are devoid of these disadvantages, in addition, their potential benefits include easy terminal drug input in case of adverse effects, permits use of drugs with a short biological half life, avoidance of absorption variability and differential metabolism associated with oral therapy ^[4]. The statical data showed a market of $ 12.7 billion in the year 2005 which is assumed to increase by $ 21.5 billion in the year 2010 and $ 31.5 billion in the year 2015. Almost all the pharmaceutical companies are developing transdermal drug delivery systems ^[5].

It is marketed in Europe for the treatment of hypertension and heart failure and is currently being reviewed for use in the US by the Food and Drug Administration. Nebivolol appears to be well tolerated with an adverse event profile that is at least similar, if not better, than that of other beta-adrenergic blockers. Studies suggest that long-term therapy with nebivolol improves left ventricular function, exercise capacity, and clinical endpoints of death and cardiovascular hospital admissions in patients with stable heart failure ^[6].

Nebivolol is a third generation beta-blocker, with highly selective for the β₁-adrenoceptors (AR) and endowed with the ability to release nitric oxide from the cardiovascular endothelium ^[7]. In animal models nebivolol has been shown to induce endothelium-dependent arterial relaxation in a dose dependent manner, by stimulation of the release of endothelial nitric oxide ^[8]. Nevibolol hydrochloride (M.W. 441.9 g:mol) showed the favourable logarithmic value of partition coefficient (Log P (octanol/water): 3.23; 4.03 (pH 11.8, 23°C). and negligible skin degradation. The plasma half life is about 8-10 hours which make frequently dosing necessary to maintain the therapeutic blood levels of drug for a long term treatment ^[9].

MATERIAL AND METHODS:

Materials

Nebivolol hydrochloride was a gifts samples from Zydus Cadila, Health care ltd., Ahmadabad (Gujarat), and Hydroxy Propyl Methyl Cellulose (HPMC) and Eudragit RS 100 were gift sample from Akums Drugs & Pharmaceutical LTD, Haridwar, Polyethylene glycol 400 (PEG 400) was purchased from Central Drug House Ltd., New Delhi and Dimethyle sulfoxide (DMSO) was purchased from Merck Specialities Pvt., Worli, Mumbai, India.

Analytical method for Nebivolol hydrochloride

A total of 10 mg of accurately weighed quantity of Nebivolol Hydrochloride was dissolved in 100 ml of methanol (concentration 100 mcg/ml). From the above stock solution 60 ml was taken and diluted with methanol to made it 100 ml to get the concentration of 60 mcg/ml. In order to generate a calibration curve, 5 to 60 μg/mL of primary standard were prepared and the calibration curve was obtained by measuring their absorbance at predetermined UV- VIS spectrophotometer at 282 nm shown in figure no.1. The volumetric flask was first rapped with black paper and then it was covered with aluminium foil to avoid the problem of drug photosensitivity. The concentration of Nebivolol hydrochloride was calculated using the linear regression equation of the calibration curve (Absorbance = 0.015 × concentration - 0.009, r2 = 0.9989). When a standard drug solution was assayed repeatedly (n = 6), mean standard error (accuracy) and RSD (precision) were found to be ±0.35 and ±0.65, respectively.

Physicochemical Compatibility of Drug and Polymer

The physicochemical compatibility between Nebivolol hydrochloride and polymers used in the films was studied by using fourier transform infrared (FTIR- 8300, Shimadzu Co., Kyoto, Japan) spectroscopy. The infrared spectra were recorded using an FTIR by the KBr pellet method and spectra were recorded in the wavelength region between 4000 and 400 cm^–1. The spectra obtained for Nebivolol hydrochloride, polymers, and physical mixtures of Nebivolol hydrochloride with polymers were compared.

Preparation of transdermal films

In the present study, drug loaded matrix type transdermal films of Nebivolol hydrochloride were prepared by solvent evaporation method ^{[10,
11, 12, 13]} using different ratios of ERS-100 and HPMC K100M polymers (Table 1). The polymers were weighed in requisite ratios by keeping the total polymer weight at 1.0 gm added in solvent mixture (3:2 ratio of chloroform, methanol). Propylene glycol was incorporated as plasticizer and DSMO as penetration enhancer were used. A known quantity of drug was added slowly to the solution and dissolved by continuous stirring for 30 min. The aluminums foil was spread uniformly on a glass petri dish and solution poured in it for the formulation of transdermal patch. The disk was kept on a horizontal surface for uniformity. The solution was poured on the foil into a petri dish of about 70 cm². The solvent was allowed to evaporate for 24 hrs by inverting a funnel over a disk. The polymer was found to be self adhesive due to the presence of Eudragit polymer along with plasticizer. The patches were cut out to give required area for evaluation.

Evaluation of transdermal patch of Nebivolol hydrochloride

Physicochemical properties such as physical appearance, thickness ^[14], weight variation ^[15], folding endurance ^{[16, 17]}, content uniformity ^[18], were determined on developed patches.

In-vitro permeation study

The in-vitro permeation study of fabricated transdermal patches of Nebivolol hydrochloride was carried out by using excised rat abdominal skin and franz diffusion cell ^[^14]. The skin was sandwiched between donor and receptor compartments of the diffusion cell. The patch of 2.64 cm² was placed in intimate contact with the stratum corneum side of the skin; the top side was covered with aluminum foil as a backing membrane. Teflon bead was placed in the receptor compartment filled with 12ml of normal saline. The cell contents were stirred with a magnetic stirrer and a temperature of 37 ± 0.5°C was maintained throughout the experiment. Samples of 2ml were withdrawn through the sampling port at different time intervals for a period of 48 h, simultaneously replacing equal volume by phosphate buffer pH 7.4 after each withdrawal. The samples were analyzed spectrophotometrically at 282 nm.

Table No.1 Composition of transdermal patches

Formulation code	Drug (mg)	Polymers ratio ERS100: HPMC K100M	DMSO	PEG 400	Solvents ratio (Methanol : Chloroform)
F1	100	2:8	20%	30%	3:2
F2	100	4:6	20%	30%	3:2
F3	100	6:4	20%	30%	3:2
F4	100	8:2	20%	30%	3:2

Table No. 2 Physiochemical evaluation of transdermal patches

Formulation code	F1	F2	F3	F4
Appearance	Thin, transparent and flexible	Thin, transparent and flexible	Thin, opaque and flexible	Thick, not flexible and opaque.
Thickness	0.263±0.67	0.289±0.55	0.301±0.61	0.219±0.75
Weight (mg)	51.01±0.80	52.15±0.68	50.5±0.75	52.02±2.15
Drug content (2.64cm²/mg)	3.75±1.08	3.87±0.98	3.61±0.13	3.67±0.28
Folding endurance	>100	>100	>100	>100

Table 3 In vitro drug permeation profile of Nebivolol hydrochloride transdermal patches

Formulation code	Zero order (R²)	First order (R²)	Higuchi (R²)	Korsmeyer-peppas (R²)
F1	0.9094	0.9956	0.9963	0.9948
F2	0.8929	0.9918	0.9878	0.9623
F3	0.8919	0.9749	0.9934	0.9790
F4	0.8655	0.9403	0.9831	0.9870

Fig.1 The UV scan of Nebivolol Hydrochloride in methanol

Fig. 2 Comparative drug permeation profile

Fig. 3. FTIR Spectra of Nebivolol Hydrochloride with polymers

Fig.4 Higuchi’s kinetic profile

Fig.5 Zero order kinetic profile

Based on the results of in-vitro permeation profiles of preliminary batches of Nebivolol hydrochloride transdermal patches the optimum composition of checkpoint batches of Nebivolol hydrochloride transdermal patch was optimized.

Stability Studies

Optimized medicated films were subjected to short term stability testing. Films were placed in a petri disk lined with aluminium foil and kept in a humidity chamber (desiccators) maintained at 40 ± 2 ⁰C and 75 ± 5% RH for 6 month as per ICH guidelines ^[19] Changes in the appearance and drug content of the stored films were investigated after storage at the end of every week. The data presented were the mean of three determinations.

RESULTS:

Evaluation of transdermal patch

The prepared transdermal patches were evaluated for their physicochemical characteristics such as appearance, weight variation, thickness, folding endurance, drug content, (Table no.2) and in vitro drug permeation through albino rat skin (Table no. 3).

The physical appearance of the various formulations in terms of their transparency, smoothness, flexibility, stickiness, homogenicity and opaque properties were recorded. The formulation F-1 was found to be thin, transparent and flexible, formulation F-2 was found to be thin, transparent and flexible, formulation F-3 was found to be thin, opaque and flexible and formulation F-4 was found to be thick, not flexible and opaque. The formulation F-1 gave the most suitable transdermal film with all desirable physico-chemical properties. The thickness of the patches was varied from 0.219 ± 0.75 mm to 0.301 ± 0.61 mm. From the result, uniformity of the patches was showed prepared by solvent evaporation while low standard deviation values ensued by thickness measurements of film. The weights ranged between 50.5 ± 0.75 mg and 52.15 ± 2.15 mg, which indicates that different batches patch weights, were relatively similar. Folding endurance was found to be >100 that is satisfactory weight of the patches, drug content was found to be 3.61 ± 0.13 mg to 3.87 ± 0.98 mg. The cumulative percentage drug permeated and percentage drug retained by the individual path in the in vitro skin permeation studies were based on the mean amount of drug present in the respective patch. The cumulative percentage drug release for F1 was found to be 91.21 ± 2.14 % at 48 h and for F4 it was found 68.16 ± 5.57 % at 24 h. The formulation, F1 [HPMC K100M, ERS-100 (8:2)] is considered as a best formulation, since it shows maximum in vitro drug release as 91.21 ± 2.14 % at 48 h showed in figure no.2.

DISCUSSION:

Trasdermal drug delivery system increases the bioavailability of drug by avoiding the first pass metabolism and increases the therapeutic efficacy of drug by reaching into the systemic circulation and also most suitable system for a long term treatment or for a multi dose treatment because transdermal patches are prepared for a long period of time in a single dose providing treatment from a day to even up to seven days. Polymers HPMC K100M and ERS-100 were selected on the basis of their adhering property and non toxicity. The result of the study showed excellent adhering property and controlled release. Result from present study concluded that Nebivolol hydrochloride in combination with HPMC K100M, ERS-100 and with incorporation of PEG 400 (30%) and DMSO (20%) produced smooth, flexible and transparent film. FT-IR studies showed characteristic peaks of Nebivolol hydrochloride, confirming the purity of the drug. FT-IR spectral studies indicated there was no interaction between Nebivolol hydrochloride and polymers used (Fig. no. 3). Nebivolol hydrochloride patches were prepared with combination of these polymers and evaluated it for physical parameters such as thickness, drug content, weight variation, % moisture loss and % moisture absorption. It was observed from this results, that thickness, weight variation, drug content, low moisture loss, low moisture absorption, tensile strength were suitable for maximum stability of the prepared formulations. The drug content of TDDS patches ranged from 3.61±0.13-3.87±0.98 mg. The drug release rate increased as the concentration of hydrophilic polymer was increased. The cumulative percentage drug release for F1 was found to be 91.21 ± 2.14 % at 48 h and for F4 it was found 68.16 ± 5.57 % at 24 h. The formulation, F1 [HPMC K100M, ERS-100 (8:2)] is considered as a best formulation, since it shows maximum in vitro drug release as 91.21 ± 2.14 % at 48 h. The drug release kinetics studies showed that the majority of formulations were governed by Higuchi model and mechanism of release was non-Fickian mediated. Higuchi developed an equation for the release of a drug from a homogeneous-polymer matrix-type delivery system that indicates the amount of drug releases is proportional to the square root of time ^[20]. When plotted against square root of time, the release of drug from the transdermal film showed a straight line, it indicates that the release pattern is obeying Higuchi’s kinetics. In our experiments, in vitro release profiles of all the different formulations of transdermal patches could be best expressed by Higuchi’s equation, for release of drug from a homogeneous-polymer matrix–type delivery system that depends mostly on diffusion characteristics.

From the in vitro permeation profile data of all the formulations through rat skin, kinetics of drug release were found for zero-order, first-order, Higuchi-type release kinetics and Korsmeyer-Peppas-type release kinetics. The coefficient of correlation (R²) of each of these release kinetics were calculated and compared (Table no.3). The data revealed that the release pattern of selected formulations was best fitted for Higuchi kinetics (Fig no.4), as the formulation coefficient values predominate over zero-order (figure no.5), first-order and Korsmeyer-Peppas type release kinetics, which again confirmed with Higuchi’s equation for the drug release from matrix. Thus, a slow and controlled release as observed is indicating that the drug release mechanism is non- Fickian model, as proposed by Higuchi.

The regression analysis of the in vitro permeation curves were carried out for in vitro permeation studies in rat skin. Among all these formulations, the formulation F-1 showed the maximum % drug cumulative release i.e. 91.21 % up to 48 hours of the study. All the formulations showed Higuchi-type release kinetics. Regression analyses of the in vitro permeation curves were carried out. The slope of the straight line obtained after plotting the mean cumulative amount released per Cm. Square patch vs. square root of time was taken as the experimental flux for Nebivolol hydrochloride. In our studies the n values calculated from the slope of the Korsmeyer-Peppas Kinetic model, which were found to be 0.54, 0.62, 0.5 and 0.52 for F-1, F-2, F-3 and F-4 patches respectively. These n values showed the release mechanism following Fick’s diffusion.

CONCLUSIONS:

In conclusion, controlled release TDDS patches of Nebivolol hydrochloride can be prepared using the polymer combinations, HPMC K100M, ERS-100 (8:2) with PEG 400 and DMSO as plasticizer and enhancer, respectively. The release rate of drug through patched increased simultaneously as concentration of hydrophilic polymer was increased. However, the mechanism of drug release of all formulations was non-Fickian. The properties of film did not change during the period of study. Further, in vivo studies have to be performed to correlate with in vitro release data for the development of suitable controlled release patches for Nebivolol hydrochloride.

ACKNOWLEDGEMENT:

Authors are grateful to Zydus Cadila health care limited, Gujarat, for providing gift samples of Nebivolol Hydrochloride and Gyan Vihar School of Pharmacy and Research Institute, Jaipur for providing necessary lab facilities.

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Received on 13.09.2012 Accepted on 21.10.2012

Asian J. Pharm. Res. 2(4): Oct. - Dec. 2012; Page 136-141