Effect of Permeation Enhancers on the Release Behavior and Permeation Kinetics of Novel Tramadol Lotions

Purpose: The aim of this research work was to formulate, characterize and evaluate the in vitro permeation behavior of tramadol lotion containing propylene glycol (PG) and polyethylene glycol (PEG) as permeation enhancers. Methods: The permeation experiments were conducted in vitro using full thickness rabbit skin in Franz diffusion cells. The donor compartment was filled with PBS (phosphate buffered saline) at pH 7.4 ± 0.1. The receptor phase was continuously stirred PBS (pH 7.4) at 37 °C ± 0.5. The amount of tramadol permeated into the receptor phase was determined spectrophotometrically at 271 nm. Various permeation parameters such as permeation coefficient (Kp), diffusion coefficient (D), flux (J), input rate, and enhancement ratio were obtained using Fick’s diffusion laws. Results: Permeation increased with increase in the concentrations of both enhancers tested. Maximum cumulative amount permeated for control lotion (L c ) was 357 µg/cm 2 /min with input rate 0.574 µg/min and lag time (t lag ) of 34.93 min, while for the optimum test lotion (L 4 , containing 8 % PG/PEG in ratio of 1:1 v/v), it was 926 µg/cm 2 /min, 1.482 µg/min and 58.36 min, respectively. The significantly (p < 0.05) higher permeability shown by the test lotion L 4 can be attributed, in part, to the interaction of PG with intercellular lipids leading to the disruption of their organization and increasing their fluidity, and also partly as a result of solubilization of lipid bilayers by PEG. Conclusion: A binary system of PG and PEG in lotion can be successfully utilized for the permeation enhancement of tramadol.


INTRODUCTION
In a broad sense, the term TDDS (transdermal drug delivery system) includes all topically administered drug formulations intended to deliver the active ingredient into general circulation [1].Ideally, the entire drug should penetrate through the skin to the underlying blood supply without any accumulation in the layers of the skin for successful transdermal delivery which often involves a demonstration of clinical safety and effectiveness.
Although TDDS was introduced more than 200 years ago [2], it is only recently that the method appears to have reached a practical stage [3].A closely related term is percutaneous delivery, which is the transport of drugs into target tissues with the aim to avoid systemic effects.The concept of percutaneous absorption of drugs was given by Stoughton in 1965 [4).TDDS provides various merits over conventional drug delivery systems such as oral and parenteral delivery, including avoidance of hepatic first pass metabolism, reduction of pain, and possible sustained release of drugs [5][6][7].
Chemical enhancers are substances which momentarily diminish skin obstacle and accelerate drug absorption.Enhancers include essential oils, terpenes, terpenoids, azone, pyrrolidones, fatty acids, sulphoxides, oxazolidinones and urea.Propylene glycol and polyethylene glycol can easily penetrate the skin and has assessed as a skin penetration enhancer in several in vitro studies [8].
Tramadol HCl (TRA) is a centrally acting opioid synthetic analgesic which acts through selective binding to the µ-opioid receptor as well as weak inhibition of norepinephrine and serotonin uptake.It is employed in severe acute or chronic pains.TRA is a basic drug with pKa = 9.3 [9,10].To the best of our knowledge, no study has addressed in detail the formulation of tramadol as a lotion.The aim of this work, therefore, was to formulate, characterize and evaluate the in vitro permeation behavior of tramadol lotion containing propylene glycol (PG) and polyethylene glycol (PEG) as permeation enhancers.

EXPERIMENTAL Chemicals
Tramadol hydrochloride (purity 99.7 %, TRA) was a gift by Ali Gohar Pharmaceuticals, Karachi, Pakistan.Propylene glycol (PG), isopropyl alcohol (IPA) and sodium chloride (NaCl) were purchased from Merck, Germany.Polyethylene glycol (PEG 1000), potassium dihydrogen phosphate and disodium hydrogen phosphate (Na 2 HPO 4 •12H 2 O) were procured from Fluka, Germany.Potassium chloride (KCl, Aldrich Chemical, USA), vacuum grease (Dow Corning, USA), carbomer-980 (Merck, Germany), sodium hydroxide (Shama Laboratory Chemical Works, Lahore) were purchased from local suppliers.All the chemicals used were of analytical grade and used without further treatment.Glass double-distilled water was used throughout the study.

Preparation of hydro-alcoholic lotions
Hydro-alcoholic lotions (L 1 -L 5 , 100 ml each) of TRA having varying concentrations (2, 4, 6, 8 and 10 %) of permeation enhancers (PG and PEG in a ratio of 1:1 v/v) were prepared in a 100 ml volumetric flask.To prepare the lotions, TRA (50 mg) was dissolved in 10 ml of isopropyl alcohol in a 100 ml volumetric flask followed by the addition of carbomer-980 (1.5 mg) with continuous shaking.PG, PEG, and phosphate buffer (0.1 ml) were added and the volume made up to 100 ml with distilled water.Control formulation (Lc) which contained the same ingredients in the same amounts, except an enhancer, was also made.

In vitro characterization studies
Each TRA-containing lotion was tested to assess its physical properties including pH, viscosity, spreadability, homogenity and drug solubility.Each of these analyses was conducted in triplicate (n = 3).
The pH of the lotions was tested using a digital pH meter (Mettler & Toledo, Giessen, Germany).The viscosity was determined using a Brookfield viscometer (Model RVTDV II, Stoughton, USA) at room temperature (25 ± 2 °C).A C-50 spindle was used with a stirring speed of 220 rpm.The gap value was fixed at 0.3 mm.Lotion spreadability was tested by the wooden block and glass slide method as presented previously [11].Briefly, 5 mL of the lotion was adjoined to a dedicated pan and the time taken for a movable upper slide to detach entirely from the fixed slides was observed.Spreadability (S) was evaluated asin Eq 1. S = M.L/t ……………………….. (1) where M = weight/volume tide to upper slide, L = length of glass slide and t = time taken to separate the slide completely from each other.Visual examination was used to evaluate the homogeneity of each lotion.To assess aggregation, the appearance of the lotions was observed [12].
Each TRA-containing lotion was tested for drug content.The lotion (5 mL) was dissolved in ethanol (5 mL) and PBS (pH 7.4) added to make up the final volume to 100 mL.The mixture was vigorously mixed for 2 h on a mechanical shaker, filtered and the supernatant layer then analyzed spectrophotometrically for TRA at 271 nm using UV/Vis spectrophotometer (model 1601, Shmadzu, Japan) [7].

Stability studies
Accelerated stability studies of the formulated lotions were conducted at a temperature of 40 ± 2 ºC, 75 %RH for 3 months.Each lotion was tested for alterations in its appearance, pH, viscosity and drug content after 12 h, 1 day, 7 days, 1 month and 3 months.Each of these analyses was conducted in triplicate (n = 3).

In vitro permeation studies
In vitro permeation studies of TRA lotions across rabbit skin were carried out using two-chambered Franz-type diffusion cells (manufactured "in house") [6] having a receptor phase of ~5 ml, and a diffusional area of ~0.788 cm  [13].Adult rabbit skin was used for permeation studies at 37 ± 0.5 o C. Abdominal full thickness skin of male White New Zealand rabbit (3 -4 kg weight) was carefully excised after sacrificing the rabbit.Subcutaneous fats and other extraneous tissues adhering to the dermis were completely removed and trimmed with forceps and scissor.The skin was cleaned with phosphate buffered saline (PBS) at pH 7.4 and stored in 500 ml normal saline in a refrigerator (18 -20 o C) [6,7].
The skin was used within one week of excision.Sheets of the skin were cut to appropriate sizes (~ 1 cm 2 in diameter) and soaked overnight in the receptor solution (PBS).The membrane was then placed between the two compartments of the diffusion cells with epidermis side facing the donor compartment while the dermal side was bathed with PBS at pH 7.4 (receptor fluid).The donor compartment was filled with PBS at pH 7.4 ± 0.1.This pH is close to that of human skin.The receptor fluid was stirred with a magnetic stirring bar at 500 rpm, keeping the temperature at 37 ± 0.5 o C by means of a water jacket.Care was exercised to remove any bubbles between the underside of the skin and the solution in the receiver compartment.Vacuum grease was used to produce a leak-proof seal between the membrane and the two compartments of the diffusion cell, i.e., donor and receptor.
The receptor and donor compartments were filled with PBS at pH 7.4 ± 0.1.To remove air bubbles and preclude the development of air pockets in the receptor phase, PBS was degassed in an ultrasonic bath.To avoid evaporation from the compartments, the cell arm and donor compartment were covered with a parafilm.Constant mixing of the receptor phase was obtained with a magnetic stirrer placed in the receptor compartment.The diffusion cells were placed on a stirring-bed immersed in a water bath at 37 ± 0.05 o C, to maintain the temperature of membrane surface.After 24 hours, both chambers were cleared of PBS and the receptor compartment was immediately refilled with prethermostated PBS, while the skin remained intact.The donor compartment was charged with 1 ml of the lotion (test formulation).At time intervals of 5, 15, 30, 60, 90, 120, 180, 240, 360 and 480 min, 0.2 ml sample was drawn, using a micro-pipette, from receptor solution followed by addition of same volume of pre-thermostated receptor solution to maintain sink conditions.The samples were analyzed spectrophotometrically at 271 nm using UV/Vis spectrophotometer (model 1601, Shmadzu, Japan) to obtain the amount of TRA permeated through rabbit skin after diluting with 1.8 ml PBS.Since skin shows great sampleto-sample permeability variations [12], each of these analyses was conducted in pentaplicate (n = 5).
To construct a calibration curve, 500 mg of TRA was dissolved in PBS (10 ml) in 100 ml volumetric flask and the final volume made up to 100 ml by adding PBS to prepare stock solution.From this solution, dilutions of 10, 20, 30, 40, 50, 60, 70, and 80 µg/ml were prepared.The resultant dilutions were analyzed spectrophotometrically for UV absorbance' maximum UV absorbance of TRA was found at 271 nm [9,10].The linear equation of the constructed calibration curve was y = 0.022x -0.021 and correlation coefficient (R 2 ) of 0.998.Steady-state flux was determined from the slope of the linear portion of the cumulative amount of permeation (Q) versus time (t) plot.The input rate of TRA (Table 1) permeating across rabbit skin was determined as in Eq 2 Input rate = K p × C × A …………….. (2) where, K p is permeability coefficient, C is donor amount (µg), i.e., amount of drug in the donor compartment and A is the Franz cell area of permeation (~0.788 cm 2 ).Enhancement ratio (ER) was calculated by dividing the flux of the test formulation by the flux of control formulation.

Statistical analysis
The results are expressed as mean ± standard deviation (SD, n = 5).Statistically significant differences between various permeation data were determined using F-test, Fisher's least significant difference (LSD), analysis of variance (ANOVA) and multiple range tests at 95 % confidence level.

Physicochemical characteristics of the tramadol lotions
All TRA-containing lotions (L 1 -L 5 ) emerged as clear, colorless and homogenous solutions without any aggregates.The pH of all the lotions was ~7.9 and there was no significant difference (p > 0.05) among all the formulations.There was, however, significant differences in their viscosities (p < 0.05), viz, (54.00 ± 0.93) ×10 -3 , (57.00 ± 0.86) × 10 , respectively.It is evident from these values that spreadability of lotion decreased with the increase in PG-PEG content.
Based on visual observation, no formulation showed any alteration in appearance, opaqueness or color at the end of the accelerated stability studies.There was also no significant difference (p > 0.05) in pH, viscosity, spreadability, drug content or permeation through rabbit skin at the end of the accelerated stability studies.

In vitro permeation
The cumulative amount permeated of TRA through rabbit skin using different hydro-alcoholic lotions is presented in Figure 1.The input rate of tramadol is shown in Table 1.The various permeation parameters calculated for the lotions are shown in Table 2. Drug permeation data (through full thickness rabbit skin) are given in Figure 1.A significant difference between the cumulative amount (µg/cm 2 ) of TRA permeated through rabbit skin in the presence of PG and PEG as permeation enhancers was found when F-Test, Fisher's least significant difference (LSD), and multiple range tests were employed, but the multiple range test did not show a significant difference between L 4 and L 5 (p > 0.05).This is also supported by the calculated enhancement ratio data shown in Table 2.
The data presented here show that PG and PEG have synergistic effects on the delivery of TRA across the skin.The results also indicate that permeation of TRA increased with increase in the concentration of PG and PEG; maximum cumulative amount of TRA permeated through rabbit skin was obtained with L 4 (Figure 1).L 1 , L 2 and L 3 showed largely the same permeation enhancement effects.

DISCUSSION
The stratum corneum (SC) of mammalian skin is considered to be a permeation barrier for water and most solutes whereas the intercellular lipid multilayer domains of SC are believed to present a diffusional pathway for most lipophilic solutes.Permeation enhancers (PG and PEG) enter the SC and change the solution properties of SC by altering the chemical environment and thus dissolve the barrier capacity of this cutaneous layer [7].
The apparent effects of the enhancers (PG and PEG) used in the present study may be dependent on their concentrations as evident   from results obtained.Permeation rate through rabbit skin followed Fick's diffusion law and was mainly affected by the amount of PG and PEG-1000 [14].The increase in permeation with increase in enhancer content (except for L 5 ) is expected based on the effect of the effect of enhancers on the skin which was mentioned above.The different behavior of L 5 may be due to very high levels of enhancers leading possibly to retention of drug in the skin [15] as indicated in Table 2.
PG also increases drug partitioning and drug permeation [16] and in combination with PEG and other enhancers, PG may increase drug flux possibly due to occupation of its hydrogen bonding sites as well as salvation of the keratin of stratum corneum.It has been suggested that the mechanism of skin permeation enhancement by PG occurs through the extraction of lipids from the SC layers [6,7,16].PEG due to its solublizing properties is considered a good skin permeation enhancer, and when other lipophilic and hydrophilic surfactants are incorporated with it in formulations, enhances solubility and reduces hepatic metabolism by forming chylomicrons that uncrease lymphatic delivery, resulting in increased bioavailability [17].This is buttressed by drug flux value of 1.271 µg/cm 2 /min for L 5 which is lower than that of L 4 due to greater solubliztion of the drug in the presence of high levels of PEG [18].
The increased permeability shown by L 4 can be attributed, in part, to the interaction of PG with intercellular lipids, leading to disruption of their structural organization and increase in their fluidity and partial solubilization of the lipiophilic matrix by PEG.While K values are in order of L 4 > L 1 > L 5 > L 2 > L 3 and D values of the lotion are in order of L 3 > L 5 > L 4 > L 2 > L 1 , thus indicating that increase in PG and PEG-1000 levels also increased the diffusivity of TRA across skin layers.

CONCLUSION
The data presented in this work suggest that formulation of an effective TDDS for the delivery of tramadol hydrochloride (TRA) is feasible.Improvement in the solubility and diffusivity of TRA is necessary to realize clinically effective transdermal delivery of the drug.Among the various combinations of PG and PEG examined in this study, formulation L 4 (containing 8 % PG/PEG 1000 in a 1:1 ratio) yielded the most promising TDDS.Further work would, however, be required to achieve clinical levels in experimental animals and humans.

Table 2 :
Computed permeation parameters for tramadol lotions