Transdermal Drug Delivery System for Atomoxetine Hydrochloride – In vitro and Ex vivo Evaluation

Mamatha T¹, Venkateswara Rao J^1*, Mukkanti K² and Ramesh G³.

¹Sultan–Ul–Uloom College of Pharmacy, Road No.3, Banjarahills, Hyderabad-500034, (A.P.), India.

²Centre for Environment, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad-500072 (A.P.), India.

³Centre for Biopharmaceutics and Pharmacokinetics, University College of Pharmaceutical Sciences, Kakatiya University, Warangal- 506 009 (A.P.), India

*For Correspondence: jvrao1963@yahoo.co.in

Current Trends in Biotechnology and Pharmacy , Volume 3 (2) April - 2009

Abstract

Monolithic matrix type transdermal drug delivery systems (TDDS) of atomoxetine hydrochloride (A-HCl) were prepared by the film casting on a mercury substrate and characterized by physicochemical characteristics like thickness, weight variation, drug content, flatness, folding endurance and in vitro drug release studies, ex vivo skin permeation studies. Eight formulations (carrying Eudragit RL100 and Hydroxypropyl methyl cellulose 15 cps in the ratios of 8:2, 6:4, 4:6, 2:8 in formulations A-1, A-2, A-3, A-4 and Eudragit RS 100 and Hydroxypropyl methyl cellulose 15 cps in the same ratios in formulations B-1, B-2, B-3, B-4 respectively) were prepared. All formulations carried 20 mg of drug, A-HCl, 10% w/w of propylene glycol as penetration enhancer, 10% w/w of dibutyl phthalate as plasticizer in ethanol. The formulations exhibited uniform thickness, weight and good uniformity in drug content. The maximum drug release in 24 hrs for A-series formulations was 95.52 % (A-3) and for B-series, it was 89.55 % (B-4). Again formulations A-3 (Kp = 3.53 X 10^-2 cm h^-1) and B-4 (Kp = 3.20 X 10^-2 cm h^-1) exhibited the best skin permeation potential in the respective series. On the basis of in vitro drug release and ex vivo skin permeation performance, formulation A-3 was found to be better than the other seven formulations. The results of the study show that A-HCl could be administered transdermally through the matrix type TDDS for effective control of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults.

Keywords: Transdermal, atomoxetine hydrochloride, propylene glycol, Eudragit, HPMC.

Introduction

Delivery of drugs into systemic circulation via skin has generated a lot of interest during the last decade as transdermal drug delivery systems (TDDS) offer many advantages over the conventional dosage forms and oral controlled release delivery systems notably avoidance of hepatic first pass metabolism, decrease in frequency of administration, reduction in gastrointestinal side effects and improves patient compliance (1). Matrix based transdermal formulations have been developed for a number of drugs such as metoprolol (2), nitrendipine (3), ephedrine (4), ketoprofen (5), propranolol (6), labetolol hydrochloride (7) and triprolidine (8).

Atomoxetine hydrochloride (A-HCl) is a potent inhibitor of the presynaptic norepinephrine transporter with minimal affinity for other monoamine transporters or receptors and is the first non-stimulant medication approved for the management of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults.

A-HCl is well absorbed after oral administration with peak plasma concentrations in 1 to 2 hours after a dose. Bioavailability is about 94% in poor metabolisers but only 63% in extensive metabolisers. Atomoxetine is metabolized primarily via the cytochrome P450 isoenzyme CYP2D6 to the active metabolite 4-hydroxyatomoxetine; a minority of the population are poor metabolisers and experience plasma concentrations about 5 times those in extensive metabolisers. The half life of atomoxetine is about 5.2 hours in extensive and 21.6 in poor metabolisers (9). A-HCl due to its low therapeutic dose (10-100 mg)color=#000000> and substantial biotransformation in liver becomes it ideal candidate for design and development of transdermal therapeutic system. A-HCl in transdermal formulations provides sustained blood levels over a prolonged period, which is required for control of ADHD.

In spite of several advantages offered by transdermal route, only a few drug molecules are administered transdermally because the formidable barrier nature of stratum corneum. Two major approaches to increase transdermal permeation rate include physical techniques (iontophoresis, electroporation, sonophoresis, and microneedles) and use of chemical penetration enhancers (PE) such as solvents, surfactants, fatty acids, and terpenes.

Propylene glycol (PG) is the most commonly used pharmaceutical excipients and have been widely employed to enhance the transdermal flux of many drugs (10-14). Various mechanisms of action have been attributed to the PG for its penetration enhancement capabilities such as increased thermodynamic activity (15), increased skin/vehicle partitioning of the drug (16), and alteration of barrier property by interacting with skin components. PG may reduce barrier property of skin by causing conformational changes either in lipid acryl chains (17) or protein domains (18) or by partial lipid extractions (19).

The objective of this study was to formulate transdermal patches of A-HCl and to evaluate the effect of PG in drug release.

Materials and Methods

Materials

Atomoxetine HCl, Eudragit RL 100 (ERL) (Rohm Pharma GmbH, Germany) and Eudragit RS 100 (ERS) (Rohm Pharma GmbH, Germany) were procured from Aurobindo Pharmaceuticals (Hyderabad, India). Liquid mercury, dibutyl phthalate (DBP), hydroxypropyl methyl cellulose (HPMC), propylene glycol (PG), disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride were purchased from S.D. Fine Chemicals Limited, India. All the materials used were of analytical grade.

Preparation of TDDS

The composition of various formulations is given in Table 1. The polymeric solution (10% w/v) was prepared by dissolving ERL-100/ ERS-100 and HPMC in different ratios, along with A-HCl, DBP and PG in ethanol. The solution was poured into a glass ring placed on the surface of liquid mercury kept in a petridish. The solvent was allowed to evaporate under ambient conditions (temperature 32C and relative humidity 45%) for 24 hours. Aluminum foil was used as backing film. The polymer was found to be self sticking due to the presence of eudragit polymers along with plasticizer. The patches were cut to give required area and stored in airtight container till further use.

Physicochemical Evaluation

Thickness and Weight Variation

The thickness of the patches was assessed at 6 different points using screw gauze. For each formulation, three randomly selected patches were used. For weight variation test, 3 films from each batch were weighed individually and the average weight was calculated (Table 2).

Flatness

Longitudinal strips were cut from each film, one from the centre and two from either side. The length of each strip was measured and the variation in the length because of uniformity in flatness was measured by determining percent constriction, considering 0 % constriction equivalent to 100% flatness (20).

% Constriction = l₁-l₂/ l₂ X 100

Where l₁ is initial length of each strip, l₂ is final length of each strip.

Folding Endurance

The folding endurance was measured manually as per the reported method (21). Briefly, a strip of the film (4 x 3 cm) was cut evenly and repeatedly folded at the same place till it broke. The thinner the film more flexible it is.

Drug Content Determination

The patch (1 cm²) was cut and added to a beaker containing 100 ml of phosphate buffered saline pH 7.4 (PBS). The medium was stirred (500 rpm) with teflon coated magnetic bead for 5 hours. The contents were filtered using whatman filter paper and the filtrate was analysed by U.V.spectrophotometer (Elico, SL-164, Hyderabad, India) at 269 nm for the drug content against the reference solution consisting of placebo films.

In vitro drug release studies

The in vitro release was carried out with the dialysis membrane using Franz diffusion cell. The cell consists of two chambers, the donor and the receptor compartment. The donor compartment was open at the top and was exposed to atmosphere. The temperature was maintained at 37 ± 0.5^o C and receptor compartment was provided with sampling port. The diffusion medium used was PBS pH 7.4 solution. The drug containing film with a support of backing membrane was kept in the donor compartment and it was separated from the receptor compartment by dialysis membrane with molecular weight cut off between 12000 to 14000 (Himedia, Mumbai, India). The dialysis membrane was previously soaked for 24 hours in PBS pH 7.4. The donor and receptor compartment hold together using clamp. The receptor compartment with 15 ml of PBS pH 7.4 was maintained at 37 ± 0.5 ^oC and stirred with magnetic capsule operated by magnetic stirrer, to prevent the formation of concentrated drug solution layer below the dialysis membrane. Samples of 3 ml, were collected at predetermined time intervals and replaced with fresh buffer. The concentration of drug was determined by UV. spectrophotometrically at 269 nm. Cumulative percentage drug released were calculated (Table 3) and plotted against time (Fig. 1 and 2). The data was fitted to different kinetic models to explain the release mechanism and pattern using the following equations.

Zero order equation Q = Q_o -kt

First order equation Q = Q_oe ^-kt

Higuchi equation Q = kt ^1/2

Where, Q is the cumulative amount of drug released, Q is the initial amount of drug, k is release constant and t is time.

Preparation of Skin

Prior approval by Institutional Animal Ethics Committee was obtained for conduction of experiment (Ref: IAEC/SUCP/03/2007). The albino rats were obtained from Sainath Animal Agency, Hyderabad, India. Albino rats weighing 170-190 gm were sacrificed using anesthetic ether. The hair of test animals was carefully removed with the help of depilatory and the full thickness skin was removed from the abdominal region. The epidermis was prepared surgically by heat separation technique (22), which involved soaking the entire abdominal skin in water at 60 °C for 45 sec, followed by careful removal of the epidermis. The epidermis was washed with water and used for ex vivo permeability studies.

Ex vivo Skin Permeation Studies

The ex vivo skin permeation studies were carried out using Franz diffusion cell (Fig. 1) with a diffusional area of 3.73 cm². Rat abdominal skin was mounted between the compartments of the diffusion cell with stratum corneum facing the donor compartment. The receiver phase is 15 ml of PBS pH 7.4, stirred at 300 rpm on a magnetic stirrer. The stratum corneum side of the skin was kept in intimate contact with the film and over that placed a backing membrane. The whole assembly was kept in a water bath at 37 ± 0.5 ^oC. Samples (3 ml) were collected at predetermined time intervals and replaced with fresh buffer. The concentration of drug was determined by U.V. spectrophotometrically at 269 nm. Cumulative percentage drug permeated was calculated and plotted against time (Fig. 3 and 4). Flux was determined directly as the slope of the curve between the steady state values of the amount of drug permeated (mg cm^-2) v/s time (hours) (23) and permeability coefficients were deduced by dividing the flux by the initial drug load (mg cm^-2) as shown in Table 3.

Results and Discussion

The results of physicochemical characteristics are depicted in Table 2. The weights are ranged from 19.1 ± 2.67 to 24.9 ± 3.76 mg and 19.6 ± 3.78 to 23.1 ± 2.90 for formulation A and B series respectively. Thickness ranged from 125 ± 1 μ to 142 ± 3 μ (A series) and 122 ± 1 μ to 134 ± 3 μ (B series). The weights are found to be high with films prepared with higher proportions of HPMC as one of two polymers. As the proportion of HPMC was decreased, the thickness was also decreased. Good uniformity in drug content was observed and it ranged from 97.1 ± 0.19 mg to 99.4 ± 0.16 mg (A series) and 97.2 ± 0.11 mg to 99.2 ± 0.19 mg (B series).

The results of flatness study showed that none of the formulations had the difference in the strip lengths before and after their cuts, thus indicating 100% flatness. It shows that no amount of constriction in the patches and thus they could maintain a smooth surface when applied onto the skin. The folding endurance was found to be between 209 ± 5.34 to 249 ± 1.00 and it was found to be satisfactory.

In vitro release studies

The results of in vitro drug release studies from transdermal patches are depicted in Fig 2 and 3. The cumulative percent of drug release from formulations of A-series was 70.70, 91.60, 95.52 and 94.4 respectively from A-1, A-2, A-3 and A-4 and of B-series was 58.02, 63.24, 85.07 and 89.55 respectively from B-1, B-2, B-3 and B-4 (Table 3). The drug release from different formulations was increased in the following order: A-3>A-4>A-2>B-4>B-3>A-1>B-2>B- 1.

Variable release profiles of A-HCl from different experimental patches composed of various blends of ERL/HPMC and ERS/HPMC were observed. The process of drug release in most controlled release devices is governed by diffusion, and the polymer matrix has a strong influence on the diffusivity as the motion of a small molecule is restricted by the three-dimensional network of polymer chains (24).

Release rates were increased when the concentration of HPMC increased in the formulations. This is because as the proportion of this polymer in the matrix increased, there was an increase in the amount of water uptake and hydration of the polymeric matrix and thus more drug was released (25 ) . Formulation A4 showed less drug release compared to formulation A3, this is because the high proportion of HPMC swellable polymer further increases the tartuosity and diffusional path length, resulted in decreased drug release. However the difference was statistically insignifinacant (p>0.05). color=#000000 size=2>

The data was fitted to different kinetic models to explain drug release mechanism. The results suggested that the drug release followed Higuchi model as it was evidenced from correlation coefficients and indicating that the drug release was taking place by the process of diffusion. The correlation coefficients (0.87 to 0.97 in A4 and A1; 0.86 to 0.98 in B4 to B1) were greater than the correlation coefficients of zero order (0.67 to 0.65 in A4 and A1; 0.68 to 0.88 in B4 to B1) and first order kinetics (0.56 to 0.71 in A4 to A1, 0.57 to 0.72 in B4 to B1). As the concentration of HPMC increases in the formulations

Ex vivo skin permeation studies

The results of ex vivo permeation of A-HCl from patches are shown in Fig 4 and 5. The cumulative percent of drug permeation from formulations of A-series was 61.94, 75.55, 84.89 and 80.97 respectively from A-1, A-2, A-3 and A-4 and of B-series was 44.4, 45.7, 62.31 and 71.45 respectively from B-1, B-2, B-3 and B-4 (Table 3). The order of drug permeation from different formulations was increased in the following order: A-3>A-4>A-2>B-4>B-3>A-1>B-2>B- 1

Formulations A-3 (84.89 %) and B-4 (71.45 %) showed maximum drug permeation in their respective series with permeability coefficients of 3.43 X10^-2 cm h^-1 and 3.02 X 10^-2 cm h^-1 (Table 3). The skin permeation profiles of the test formulations were in conformity to the in vitro drug release pattern. The cumulative amount of drug permeated as well as the permeability coefficient (Kp) for TDDS were in the order of A-3>A-4>A-2>A-1 and B-4>B-3>B-2>B-1 for the A and B series, respectively. The results corroborated that higher the drug release from the formulation, higher was the rate and extent of drug permeation. Again the Kp for formulation A-3 was high than B-4 leading to conclusion that ERL 100 and HPMC combination is better than ERS 100 and HPMC as the polymeric precursor for the A-HCl transdermal formulation. As the concentration of hydrophilic polymer was increased, the amount of drug permeated was increased. This may be a result of the initial rapid dissolution of the hydrophilic polymers when the patch is in contact with the hydrated skin, which results in accumulation of high amounts of drug on the skin surface and thus leads to the saturation of the skin with drug molecules at all times (26). Drug release rate from films containing higher proportions of lipophilic polymer ERL 100 and ERS 100 may be contributed to the relatively hydrophobic nature of polymer which has less affinity for water. This results in decrease in the thermodynamic activity of the drug in the film and decreased drug permeation.

Comparison between the best formulations of respective series (A-3 and B-4) revealed that extent of drug release was higher in case of A-3 (polymers ERL 100 and HPMC) than B-4 (polymers ERS 100 and HPMC). The maximum drug permeation from formulation A-3 might be due to higher permeability characteristics of ERL 100 in comparison to ERS 100. The formulation A-3 showed an increase in permeation than the A-4 may be due to decreased in path length to the movement of drug, as it is inversely proportional to diffusion rate.

Any vehicle can have three models of penetration enhancement that is by changing thermodynamic activity or by improving skin/ vehicle partition coefficient or by altering the barrier property of stratum corneum.

Propylene glycol (PG) action as a sorption promoter has been explained in the literature on the basis of its co solvency effect. Where thermodynamic activity is considered as main driving force and also by carrier mechanism, in which PG partition into the skin and thereby promotes the movement of the drug into and through the skin. PG shows penetration enhancement activity towards 5-fluorouracil (27), progesterone (28) and estradiol (29).

Conclusions

Ex vivo permeation of A-HCl shows that patches of ERL 100:HPMC is suitable compared to ERS 100:HPMC patches. The results of the study show that A-HCl could be administered transdermally through the matrix type TDDS for effective control of ADHD. Further work is recommended in support of its efficacy by long term pharmacokinetics and pharmacodynamic studies on human beings.

Acknowledgements: The authors are grateful to the management of the institute, Sultan-Ul-Uloom Educational Society, Banjarahills, Hyderabad for providing the facilities. The gift samples of Atomoxetine HCl, Eudragit RL 100 and Eudragit RS100 by Aurobindo Pharmaceuticals, Hyderabad, India is highly acknowledged.

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Table 1. Composition of Atomoxetine HCl Transdermal Delivery Systems

Note: All the formulations carried 10 % w/w propylene glycol as penetration enhancer

All the formulations carried 10 % w/w dibutyl phthalate as plasticizer.

Table 2. Physicochemical Characteristics of Prepared Films

^a Values presented are mean ± S.D (n=3 )

Table 3. In vitro drug release and skin permeation of the developed TDDS

^a Values presented are mean ± S.D (n=3 )

Fig. 1. Schematic diagram of Franz diffusion cell

Fig. 2. In vitro release profiles of atomoxetine hydrochloride from TDDS using Franz diffusion cell.

Fig. 3. In vitro release profiles of atomoxetine hydrochloride from TDDS using Franz diffusion cell.

Fig. 4. Ex vivo permeation profiles of atomoxetine hydrochloride from TDDS using Franz diffusion cell.

Fig. 5. Ex vivo permeation profiles of atomoxetine hydrochloride from TDDS using Franz diffusion cell.