Investigation of Nanoemulsion System for Transdermal Delivery of Domperidone: Ex-vivo and in vivo...

Current Nanoscience, 2008, 4, 381-390 381

1573-4137/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd.

Investigation of Nanoemulsion System for Transdermal Delivery of Domperidone:

Ex-vivo and in vivo Studies

Sohail Akhter, Gaurav K. Jain, Farhan J. Ahmad, Roop K. Khar, Neelu Jain, Zeenat I. Khan and Sushama Talegaonkar*

Dept of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi-62, India

Abstract: The aim of the present study was to investigate the nanoemulsion system for enhanced percutaneous penetration of domperi-

done. Pseudoternary phase diagrams were constructed in order to optimize the surfactant, cosurfactant and surfactant: cosurfactant weight ratios (Smix). Nine nanoemulsion formulations were selected, characterized and their ex-vivo permeation studies using rat skin were per-

formed. The nanoemulsion formulations had small droplet size (<90 nm), uniform size distribution (PI, < 0.2) and low viscosity (<160 mP). The results demonstrated that the droplet size and viscosity of nanoemulsion decreased following decrease in the concentration of

polysorbate 20, whereas transdermal flux was increased. The optimized formulation NE-B1, which contained oleic acid (4 % w/w), polysorbate 20 (10 % w/w), diethylene glycol monoethyl ether (20 % w/w) and water (64 % w/w) showed significant increase (P < 0.01)

in the transdermal flux (169.32 ± 8.33 g.h-1cm-2). The in vivo studies revealed a 3.5 fold increase in relative bioavailability through transdermal application of NE-B1 formulation compared to oral drug suspension. Moreover, the effective drug plasma concentration was

maintained for 16 hour after the transdermal application indicated that the developed nanoemulsion systems could be a promising carrier for the transdermal delivery of domperidone for prolonged period.

Key Words: Nanoemulsions, transdermal delivery, pseudo ternary phase diagrams, domperidone, steady state flux, oleic acid.

1. INTRODUCTION

Microemulsions are quaternary systems composed of an oil phase, a water phase, surfactant frequently in combination with cosurfactant [1-3]. These spontaneously formed systems possess specific physicochemical properties such as transparency, optical isotropy, low viscosity and thermodynamic stability. In stable mi-croemulsion, droplet diameter is usually within the range of 10-100 nm (100–1000 A°), and therefore these systems are also termed as nanoemulsions (NE) [4]. Due to their unique physicochemical properties, NE offer advantages over traditional topical and trans-dermal drug delivery formulations. Many studies have shown that NE formulations possess improved transdermal and dermal delivery properties both in vitro [5-17], as well as in vivo [18-22]. The high solubilizing capacity of NE enables them to increase the solubility of poorly water-soluble drugs. Both, increase in solute concentra-tion and the tendency of the drug to favor partitioning into the stra-tum corneum make NE a useful vehicle to enhance transdermal drug permeability [23].

As demonstrated by a recent publication [18], the transdermal permeation rate of a lipophilic drug significantly increased from NE as compared to macroemulsions. In macroemulsions the free mobil-ity of the active material between the internal (disperse) phase to the external (continuous) phase within the structure of the formu-lated system is limited due to the strong interactions between the surfactants that form tight interfacial film. In NE, the co-surfactant lowers the interfacial tension of the surfactant film, resulting in a more flexible and dynamic layer [3,14]. The drug in this energy-rich system can diffuse across the flexible interfacial surfactant film, a thermodynamic process that increases partitioning and diffu-sion into the stratum corneum.

This article is intended to demonstrate the feasibility of new o/w NE system for transdermal delivery of domperidone. Domperi-done is a dopamine (D2 receptor) antagonist with predominant peripheral activity. It is clinically effective in conditions such as diabetic gastroparesis, chronic dyspepsia, pediatric vomiting, cancer chemotherapy induced nausea and emesis [24]. Owing to its high portion of hepatic first pass metabolism (~85 %) [25], its low mo-lecular weight (426 Da), its moderate lipophilicity (Log P, 3.9) as

*Address correspondence to this author at the Dept of Pharmaceutics, Faculty of Phar-macy, Jamia Hamdard, New Delhi-62, India; E-mails: [email protected], [email protected]

well as its clinical effectiveness in low doses (6mg to 20mg) [26,27], the topical application of domperidone provides, therefore, a preferred alternative to the oral dosage form.

Transdermal administration of domperidone might offer some advantages over oral route in subjects with cancer chemotherapy and those suffering from motion sickness. In particular, the trans-dermal route might (i) allow administration of lower doses through avoidance of first pass metabolism, (ii) provide sustain and constant plasma levels, (iii) improve the patients compliance and (iv) reduce frequency of administration. Previously, attempts have been made to increase the skin permeation of domperidone through modifica-tion of skin barrier properties and/or providing a driving force for drug transport [28, 29]. Despite the use of a permeation enhancer the steady state fluxes were considered too low to reach the thera-peutic level (~18 ng/mL) and the lag time remained very high [28]. In a more recent in vitro study using rat skin, rapid delivery of domperidone has been achieved by application of both iontophore-sis and electroporation, however, domperidone flux is not improved [29].

In transdermal delivery, the goal of dosage design is to maxi-mize the flux through the skin into the systemic circulation. The NE system is a promising vehicle due to powerful ability to deliver drug through skins [13]. Therefore, the transdermal delivery of domperidone using NE system is expected to provide high trans-dermal flux. Therefore the present study focuses on ex- vivo and in vivo evaluation of domperidone loaded o/w NE without using an additional penetration enhancer. The stable NE system consisted of oleic acid, commonly used non ionic surfactant (polysorbate 20), non irritant cosurfactant (diethylene glycol monoethyl ether) and water was prepared, its physicochemical properties and transdermal permeation ability of domperidone were evaluated. Furthermore, for the optimized formulation in vivo studies and skin irritation test were also conducted.

2. MATERIALS AND METHODS

2.1. Materials

Domperidone was obtained from Torrent Pharmaceuticals (Ahmedabad, India). Polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), oleic acid, isopropyl myristate (IPM), olive oil, glyc-erol triacetate (Triacetin), castor oil, Octylphenoxy polyethoxy ethanol (triton x 100), poly ethylene glycol 200 (PEG 200), poly ethylene glycol 400 (PEG 400), propylene glycol, sorbitan mono-laurate (Span 20), diethylene glycol monoethyl ether (Carbitol),

382 Current Nanoscience, 2008, Vol. 4, No. 4 Akhter et al.

soyabean oil and coconut oil were purchased from CDH (Delhi, India). Oleolyl macrogolglyceride (Labrafil M), polyethylene gly-col dicaprylocaprate (Labrafac), caprylocaproyl macrogol-8-glyceride (Labrasol) and propyleneglycol mono laurate (Laurogly-col 90) were obtained from Gattefosse (Saint-Priest, France). Poly-ethoxylated castor oil (Cremophore EL) was purchased from Sigma (Mumbai, India) whereas propylene glycol mono caprylic ester (Safsol 218) was obtained as a gift sample from Nikko chemicals (Tokyo, Japan). High-performance liquid chromatography (HPLC) grade solvents were obtained from Merck (Mumbai, India). All other chemicals used in the study were of analytical grade.

2.2. HPLC Analysis of Domperidone in Samples from Receptor

Solutions and Plasma

Domperidone was analyzed by reverse phase HPLC column (Supelco column 516, C18, 5 m, 250 mm x 4.6 mm). The HPLC system (SCL-10 AVP, Shimadzu, Japan) consisted of a binary pump (LC-10 ATVP Shimadzu, Japan), and a UV-vis detector (SPD-10 AVP, Shimadzu, Japan). The mobile phase was methanol: water (70:30 v/v) with a flow rate of 1.5 mL/min. The detection wavelength was set at 287 nm and the retention time was 6.9 min. The assay was linear (r

2 = 0.9995) in the concentration range of

0.01–50 μg/mL with a lowest detection limit at 0.005 μg/mL. The percentage recoveries ranged from 98.0 % to 101.0 %. No interfer-ence of all the formulation components was observed.

For the analysis of samples from receptor solution, aliquots of 20 l from each sample were injected via the manual injector into a HPLC system, whereas for analysis of plasma samples, 1.0 mL of plasma was taken with 2.0 mL of methanol, vortexed and centri-fuged for 15 min at 10,000 rpm. The supernatant was taken, evapo-rated to dryness and the residue was reconstituted with mobile phase. Aliquots of 20 l from each sample were injected via the manual injector. All samples were filtered through 0.45 m pore size membrane filter before injection.

2.3. Preparation of Nanoemulsions

2.3.1. Determination of Solubility of Domperidone in Oils, Sur-factants and Cosurfactants

To find out the suitable oil which can be used as the oil phase in NE and provide excellent skin permeation rate of domperidone, the solubility of domperidone in various oils (oleic acid, IPM, olive oil, glycerol triacetate, castor oil, propylene glycol mono caprylic ester, polyethylene glycol dicaprylocaprate, coconut oil, soyabean oil) was measured. The solubility of domperidone in various surfactants (polysorbate 20, polysorbate 80, polyethoxylated castor oil, capry-locaproyl macrogol-8-glyceride, sorbitan monolaurate, triton x 100, propyleneglycol mono laurate and oleolyl macrogolglyceride) and cosurfactants (diethylene glycol monoethyl ether, PEG 200, PEG 400 and propylene glycol) was also determined. An excess amount of domperidone was added in 2.0 mL of the selected oil, surfactant and cosurfactant in stoppered vials (capacity 5.0 mL) and then pre-liminary mixing was carried out over magnetic stirrer for few minuets. Later on, these vials were kept in mechanical bath shaker for 72 hours at 37 ± 0.5

oC. The equilibrated samples were centri-

fuged at 10,000 rpm for 10 min. The supernatant was separated, filtered and after appropriate dilution with methanol, solubility was determined by validated HPLC method at 287 nm as described in section 2.2.

2.3.2. Construction of Pseudo-Ternary Phase Diagram

In order to optimize surfactant and cosurfactant, phase diagrams for each surfactant and cosurfactant combinations were constructed by using aqueous titration method. The ratio of each of selected surfactant to cosurfactant (Smix) was kept constant (1:1) while oil to Smix ratio was taken 1:9. Surfactant and cosurfactant were se-lected on the basis of the number of NE points demonstrated by

phase diagrams. After selection of surfactant and cosurfactant, their optimum concentration ranges were determined by detailed study of phase diagrams using different ratios of Smix (1:0, 1:1, 2:1, 3:1, 4:1, 1:2, 1:3, 1:4). For each Smix ratio, oil: Smix ratio was varied. Total sixteen different combinations of oil and Smix (1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3.5, 1:3, 1:2.3, 1:2, 1:1.5, 1:1, 1:0.7, 1:0.43, 1:0.25, 1:0.1) were made so that maximum ratios was covered for the study to delineate the boundaries of phases precisely formed in the phase diagrams. Slow titration with aqueous phase was performed to each weight ratio of oil and Smix and visual observation were carried out for transparency, flowability and physical state of NE. For prepara-tion of drug loaded NE, domperidone (6 mg/mL) was added to the oil phase of the NE.

2.4. Characterization of Nanoemulsions

2.4.1. Transmission Electron Microscopy (TEM)

Morphology of the NE was studied using TEM (Morgagni 268D SEI, USA) operating at 200 KV and of a 0.18 nm capable of point to point resolution. Combination of bright field imaging at increasing magnification and of diffraction modes was used to re-veal the form and size of the NE. In order to perform the TEM ob-servations, the diluted NE was deposited on the holey film grid and observed after drying.

2.4.2. Droplet Size and Size Distribution

Droplet size was determined by photon correlation spectros-copy that analyzed the fluctuations in light scattering due to brownian motion of the particles [30], using a Zetasizer (1000 HS, Malvern Instruments, U.K). The formulation (0.1 mL) was dis-persed in 50 mL of water in a volumetric flask, mixed thoroughly with vigorous shaking and light scattering was monitored at 25°C at a 90° angle. Polydispersity index (PI), for the formulations was determined as ratio of standard deviation to the mean droplet size of the formulation.

2.4.3. Viscosity

The viscosity of the formulations was determined using Brookfield DV III ultra V6.0 RV cone and plate rheometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) using spindle # CPE40 at 25 ± 0.5 °C. The software used for the calcula-tions was Rheocalc V2.6.

2.4.4. Refractive Index and pH

Refractive index of NE was determined using an Abbes type re-fractometer (Nirmal International, New Delhi, India) at 25 ± 0.5 °C. The apparent pH of the formulation was measured by pH meter (AccumentAB 15, Fisher scientific, U.S.A.) in triplicate at 25 ± 1ºC.

2.5. Thermodynamic Stability of Nanoemulsions

To assess the thermodynamic stability of drug loaded NE, clar-ity, phase separation, droplet size and drug content were evaluated before and after subjecting to following stress tests as previously reported [31]:

• Heating cooling cycle: NE formulations were subjected to six cycles between refrigerator temperature (4°C) and 45°C (sto-rage not less than 48 h at each temperature). Stable formula-tions were then subjected to centrifugation test.

• Centrifugation: Formulations were centrifuged at 3500 rpm for 30 min and those that did not show any phase separation were taken for the freeze thaw stress test.

• Freeze thaw cycle: Formulations which passed centrifugation test, were subjected to three freeze thaw cycles between

21 °C and +25 °C (storage not less than 48 h at each temperature).

Investigation of Nanoemulsion System for Transdermal Delivery Current Nanoscience, 2008, Vol. 4, No. 4 383

2.6. Ex-vivo Permeation Studies

2.6.1. Preparation of Rat Skin

The male albino wistar rats (7-9 weeks old, 200-250 g) were sacrified by aspiration of ethyl ether and the abdominal skin was carefully excised from the underlying connecting tissue using scal-pel. The hairs remaining on the skin were trimmed away. The sub-cutaneous tissue was removed surgically and the dermis side was wiped with isopropyl alcohol to remove the adhering fat. The cleaned skin was washed with distilled water and stored at 21°C until further use.

2.6.2. Skin Permeation Study

The ex-vivo skin permeation studies were performed using Franz diffusion cell apparatus (FDC-6, LOGAN Instrument Corp., Somerset, NJ, USA). The diffusion area was 0.75 cm

2 and receptor

volume was 5.0 mL. The skin was brought to room temperature, cut and trimmed to appropriate size and mounted between the donor and receptor compartments of the diffusion cell with the stratum corneum side facing upwards and then the donor chambers were clamped in place. The receptor compartment was filled with di-methyl formamide and phosphate buffer saline (pH 7.4) in the ratio 1:9 v/v. The dimethyl formamide was added into receptor com-partment in order to maintain the sink conditions. The receptor fluid was stirred with a magnetic rotor at a speed of 600 rpm and the temperature was maintained at 37 ± 1°C. After stabilization of the skin (when the receptor fluid showed negligible signal on HPLC detector), one mL of NE formulations, was placed into the donor compartment and sealed with paraffin film to provide occlusive conditions. Samples were withdrawn at regular intervals (1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 h), filtered through 0.45- m membrane filter and analyzed for drug content by HPLC method as described in section 2.2. The receptor phase was immediately replaced with equal volume of fresh receptor fluid. The same study was also per-formed for control formulations. Each set of experiment was per-formed with 3 diffusion cells (n=3).

2.6.3. Calculation of Permeation Parameters

The cumulative amount of domperidone permeated per unit of rat skin surface area, Qt/S (S=0.75 cm

2) was plotted as a function of

time (t, h). The permeation rate of domperidone at steady-state (Jss, g/cm

2/h) through rat skin was calculated by linear regression in-

terpolation of the cumulative amount permeated through rat skin per unit area vs time:

Jss = Qt / S. t (Eq. 1)

The permeability coefficient (Kp, cm/h) was calculated accord-ing to the equation:

Kp = Jss / Cd (Eq. 2)

Where Cd = concentration of drug in donor compartment (6.0mg/mL), and is assumed that under sink conditions the drug concentration in the receiver compartment is negligible compared to that in the donor compartment.

The enhancement ratio (ER) was calculated according to the equation:

ER = Flux from formulation / flux from formulation E. (Eq.3)

All skin permeation experiments were repeated three times and data were expressed as mean of three experiments ± standard devia-tion (S.D).

2.7. In vivo Permeation Study

The animal protocol to carry out in vivo study and skin penetra-tion study was reviewed and approved by Institutional Animal Eth-ics Committee, Jamia Hamdard (approval no: 253) and their guide-lines were followed for the studies. Male albino rats, 7-9 weeks old and weighing 200–250 g were used for the study. The animals were

kept under standard laboratory conditions (temperature: 25 ± 2 °C; relative humidity: 55 ± 5%). The animals were housed in polypro-pylene cages, with free access to standard laboratory diet (Lipton feed, Mumbai, India) and water ad libitum. For in vivo permeation study, two groups, each containing eight rats were formed. To con-trol group, one mL of 0.6 % domperidone suspension (6.0 mg/mL; prepared by suspending Domestal® 10mg tablet in aqueous media) was administered orally using oral feeding sonde. For transdermal application (test group), the rats were anesthetized by i.v. injection of a combination of ketamine hydrochloride (75 mg/kg) and xy-lazine (5 mg/kg). The hair on abdominal skin was trimmed off and the abdomen was washed gently with distilled water. The drug loaded (6.0 mg/mL) optimized NE formulation was then applied to the skin surface (2.0 cm

2) in open containers glued to the skin by a

silicon rubber. The blood samples (0.2 mL) of both control and test groups were collected at predetermined time intervals through the tail vein of rat in vacutainer tubes, mixed and centrifuged at 5000 rpm for 20 min. The plasma was separated and stored at

21 °C until drug analysis was carried out using HPLC method as described under section 2.2. Pharmacokinetic parameters (PK) were calculated by noncompartmental analysis also called as model in-dependent analysis using WinNonLin version 4.0 (Pharsight Corp., Mountain View, CA). Peak plasma concentration (Cmax) and time of its occurrence (tmax) were read directly from the individual plasma concentration–time profiles. Area under concentration time curve (AUC0 t) was calculated according to linear trapezoidal method whereas mean residence time (MRT) was calculated by dividing the AUMC0 t by AUC0 t.

2.8. Statistical Analysis

Data of in vivo analysis was expressed as mean of eight animal ± S.D. The data was compared for statistical significance by the one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons test using GraphPad Instat software (GraphPad Software Inc., CA, USA).

2.9. Skin Irritation Test

The skin irritation study was performed using eight rats. A sin-gle dose of 10 μL of the formulation was applied to the left ear of the rat and the right was considered as a control. The development of erythema was monitored for six days using the method, as re-ported by Abbe et al. [32].

3. RESULT AND DISCUSSION

3.1. Screening of Oil

Being a moderately lipophilic drug, it was very important to

find out an appropriate solvent to dissolve domperidone, because

only the dissolved drug can permeate through skin. In order to

screen appropriate solvent/s for the preparation of NE, the solubility of domperidone in various oils, surfactants and cosurfactants was

measured.

Solubility of domperidone in oleic acid was found to be

200±0.012 mg/mL, which was the best among the oils investigated.

In addition, oleic acid itself is a known permeation enhancer

[33,34] and was selected as an oil phase in the present study. Based

on preliminary solubility studies, the surfactants; polysorbate 20

(3.00 ±0.39 mg/mL), polysorbate 80 (2.80 ±0.35 mg/mL), poly-ethoxylated castor oil (3.40 ±0.41 mg/mL) and caprylocaproyl mac-

rogol-8-glyceride (7.50 ±0.70 mg/mL) and cosurfactants; diethyl-

ene glycol monoethyl ether (3.50 ±0.41 mg/mL), PEG 200 (5.00

±0.45 mg/mL), PEG 400 (2.90 ±0.39 mg/mL) and propylene glycol

(3.50 ±0.40 mg/mL) showing comparable solubility of drug, were

chosen for further optimization of NE formulations. In the present

study, the selection of surfactant and cosurfactant was based on

formation of larger NE region in the pseudo ternary phase diagram


since oleic acid (oil phase) dissolved the maximum dose of the drug

easily. Constructed pseudo-ternary phase diagrams are self explana-

tory about the presence of NE region which assists easy selection of

ingredients proportions for preparation of stable formulation. Large

NE region would also facilitate the selection of formulation with

low surfactant and cosurfactant concentration, desirable for prepar-

ing non irritating formulations.

3.2. Screening of Surfactant/Cosurfactant

Aqueous titrations were done by taking polysorbate 20, polysorbate 80, caprylocaproyl macrogol-8-glyceride and poly-ethoxylated castor oil as surfactant with different cosurfactants: diethylene glycol monoethyl ether, PEG 200, PEG 400 and propyl-ene glycol. Sixteen possible combination were made and in each combination a constant surfactant to cosurfactant ratio (1:1) was taken, while oil to Smix ratio was kept at 1:9 since higher concen-tration of Smix is favorable for maximum NE formation [4,31].

Each combination was titrated with water and the resultant physical state of NE was marked on a pseudo ternary phase diagram with one axis representing the water, one representing oil and the third representing a Smix. Number of NE points was counted and results are shown in Table 1. After studying the results, it was found that the combination consisted of polysorbate 20 as a surfactant, and diethylene glycol monoethyl ether as a cosurfactant, was promising due to maximum NE region represented as points (Table 1).

3.3. Phase Diagram Study

Physical appearance of all NE formulations showed no distinct

conversion boundaries from w/o to o/w at all Smix ratios. The rest

of the region on the phase diagram represents the turbid and con-

ventional emulsions based on visual observation. Significant differ-ence was seen in ternary phase diagrams of NE constructed with

different Smix ratio (Fig. 1a-1g).

Table 1. Summarized Results of Number of Nanoemulsion Points Obtained by Different Surfactants/Cosurfactants (Smix) with Oleic Acid (Oil

Phase)

Number of Nanoemulsion points a,b

Surfactants

Cosurfactants Polysorbate 20 Polysorbate 80

Caprylocaproyl

macrogol-8-glyceride

Polyethoxylated

Castor Oil

DGME 5 2 0 2

PEG 200 2 1 0 1

PEG 400 2 0 0 1

PG 0 0 0 0

aat Smix ratio 1:1 and oil to Smix ratio 1:9; bAs obtained from respective phase diagrams; DGME: diethylene glycol monoethyl ether; PEG: poly ethylene glycol; PG: propylene glycol

Fig. (1). Pseudo-ternary phase diagrams of nanoemulsion region of existence (represented by dots) with different Smix (surfactant/ cosurfactant ratio; (a) 1:0,

(b) 1:1, (c) 1:2, (d) 1:3, (e) 2:1, (f) 3:1, (g) 4:1) composed of oleic acid, polysorbate 20 (surfactant), diethylene glycol monoethyl ether (cosurfactant) and water (% w/w).

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It was observed, when polysorbate 20 was used alone without

diethylene glycol monoethyl ether (Smix ratio 1:0), very low

amount of oleic acid (12% w/w) could be solubilized at high con-

centration (55% w/w) of polysorbate 20 (Fig. 1a). This could be

attributed to the fact that transient negative interfacial tension and

fluid interfacial film is rarely achieved by the use of single surfac-

tant, usually necessitating the addition of a cosurfactant [3,35].

At equal amounts of polysorbate 20 and diethylene glycol monoethyl ether (Smix ratio 1:1), the NE region in the phase dia-

gram increased significantly (Fig. 1b) compared to that obtained at

Smix ratio 1:0 (Fig. 1a). The presence of diethylene glycol

monoethyl ether (cosurfactant) decreases the bending stress of in-

terface and makes the interfacial film sufficiently flexible to take up

different curvatures required to form NE over a wide range of com-

positions [3,36]. However, when concentration of diethylene glycol

monoethyl ether with respect to polysorbate 20 was increased

(Smix ratio 1:2 and 1:3) the NE area was decreased (Fig. 1c and 1d,

respectively) compared to Smix ratio 1:1 (Fig. 1b). The decrease in

the NE area is possibly due to presence of low concentration of

polysorbate 20 which reduces the amount of micelles and conse-quently decreases the solubilization capacity of NE [15]. Moreover,

NEs formed at Smix ratio 1:3 were unstable and showed phase

separation within 24 h (data not shown). With further increase in

diethylene glycol monoethyl ether concentration (Smix ratio 1:4),

not a single NE point was found. In contrast to this, when concen-

tration of diethylene glycol monoethyl ether with respect to

polysorbate 20 was decreased (Smix ratio 2:1), the NE area was

increased (Fig. 1e). compared to Smix ratio 1:1 (Fig. 1b). However,

at further lower diethylene glycol monoethyl ether concentrations

(Smix ratio 3:1 and 4:1), the NE area was decreased (Fig. 1f and 1g,

respectively), it was because diethylene glycol monoethyl ether is a

polar solvent with the tendency to highly incorporate into water, and the relatively lower diethylene glycol monoethyl ether content

in the NE system decreases the hydrophilicity of the Smix, so the

area of o/w NE was decreased. In brief, system at Smix ratio 2:1

formed large isotropic NE region than the systems at other Smix

ratios.

3.4. Selection of Formulations from Phase Diagrams

Following criteria were chosen for the selection of formula-

tions:

• The oil concentration should be such that it solubilizes the single dose of the drug. Six milligram of domperidone will ea-sily be dissolved in 30 L (3% of 1 mL) of oil.

• Formulations with Smix ratio 1:0 and 1:3 were not selected because the formulations were unstable and showed phase se-paration. Furthermore, at Smix ratio 1:3, none of the formula-tions (Fig. 1d) could solubilize the amount of oil necessary to carry the single dose of the drug.

• Formulations with Smix ratio 3:1 and 4:1 were not selected because of the presence of high concentration of surfactant which might cause skin irritation. Moreover at these Smix ra-tios the area of NE isotropic region was comparatively small.

Based on the phase diagrams, three Smix ratios 1:1 (NE-A), 1:2 (NE-B) and 2:1 (NE-C) were optimized. From the selected Smix ratios, NE compositions with 30 % (NE-A1, NE-B1, NE-C1), 35 % (NE-A2, NE-B2, NE-C2) and 40 % (NE-A3, NE-B3, NE-C3) Smix ratios were selected from the region of existence (Table 2).

Fig. (2). Microphotograph of domperidone loaded nanoemulsion by TEM

(100,000 ).

3.5. Characterization of Nanoemulsions

In this work, the influence of concentration of NE components and domperidone on the characteristics of NE was studied The TEM analysis reveled that NEs droplets (placebo and drug loaded) were spherical in shape, discrete with size in nanometer range(< 100nm) ( Fig. 2).

Table 2. Composition of Selected Nanoemulsion Formulations

Percent w/w of Components in Formulation Smix Ratio

a Formulation Code

b

Oil (%) Water (%) Smix (S + CoS) (%)

NE-A1 3.0 67.0 30.0 (15.0 + 15.0)

NE-A2 5.0 60.0 35.0 (17.5 + 17.5) Formulation NE-A

Smix ratio = 1:1

NE-A3 7.0 53.0 40.0 (20.0 + 20.0)

NE-B1 4.0 64.0 30.0 (10.0 + 20.0)

NE-B2 5.0 60.0 35.0 (11.7 + 23.3) Formulation NE-B

Smix ratio = 1:2

NE-B3 6.0 54.0 40.0 (13.3 + 26.7)

NE-C1 4.0 66.0 30.0 (20.0 + 10.0)

NE-C2 5.0 60.0 35.0 (23.3 + 11.7) Formulation NE-C

Smix ratio = 2:1

NE-C3 7.0 53.0 40.0 (26.7 + 13.3)

a Surfactant/cosurfactant ratio; b NE represents nanoemulsion; A, B and C represents Smix ratio 1:1, 1:2 and 2:1, respectively; Suffix 1,2 and 3 represents Smix concentration 30, 35 and 40 %, respectively.

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Values of polydispersity index (PI), which is a measure of uniformity of droplet size within the formulation, were also calculated. All the NE formulations exhibited a narrow size distribution (PI < 0.181) (Table 3). The results of particle size analysis (Table 3) were in agreement with the droplet size measured by TEM photograph (Fig. 2). The mean droplet size of drug free NE-B formulations ranges from 32.0 to 36.10 nm, which was lower as compared to the drug free NE-A (38.02 to 47.02 nm) and NE-C formulations (47.40 to 58.55 nm) (Table 3). It is hypothetically described that at the optimum Smix ratio (1:2 for present study) the diethylene glycol monoethyl ether was exactly inserted into the cavities between the polysorbate 20 molecules, causing the interfacial film to condense and stabilize, resulting in smallest droplet diameters (32.0 ± 2.88 nm) with lowest polydispersity value (0.125 ± 0.05). In addition, particle size analysis revealed that with increase in oil concentration the droplet size of NE increases, irrespective of the Smix ratio. Large droplet size of oil rich formulations could be attributed to the fact that the higher concentration of Smix is required to solubilize the oil phase in the aqueous phase resulting an increase in size [37]. As a result of viscosity measurements, a similar behavior, as for droplet size was obtained. Viscosity of all the NE formulations was very low as expected for o/w emulsion [38] (Table 3). When formulations with different Smix ratios were compared, the minimum viscosity values were obtained for NE-B formulations (28.55 ± 2.03 to 52.70 ± 2.90 mP). The low viscosity may be due to the presence of low amount of polysorbate 20 (a fatty acid polyhydric alcohol ester having high intrinsic viscosity) compared to diethylene glycol monoethyl ether (a short chain alcohol having low intrinsic viscosity) [3]. However, for NE-A and NE-C formulations, a substantial increase in surfactant proportions resulted in higher viscosity values (Table 3).

Data also demonstrated that the viscosity values were directly related to the concentration of oleic acid, and within each Smix ratios, formulations with lowest concentration of oleic acid (NE-A1, NE-B1 and NE-C1) have lowest viscosity values (64.00 ± 3.40, 28.55 ± 2.03 and 100.0 ± 3.40 mP, respectively) as compared to other formulations. When observing the size and viscosity values of all the NE formulations (Table 3), a correlation between the droplet size and viscosity with respect to concentration of polysorbate 20 in the NE formulations was established (Fig. 3). Fig. (3) shows that the droplet size and viscosity values increased following an increase

in the amount of polysorbate 20 in the NEs, having a good correla-tion, ie, r

2 = 0.9924 and 0.9939 for droplet size and viscosity, re-

spectively.

All NE formulations studied maintained NE characteristics after addition of domperidone (6.0 mg/mL). However, the droplet size of drug loaded NE formulations was approximately 1.5 times higher than the corresponding drug free NE formulations (Table 3). One can conclude that domperidone accumulated in the interfacial layers rather than staying in the continuous phase. In contrast to results obtained for droplet size, the incorporation of drug did not signifi-cantly affect the viscosity values of NEs (Table 3). Refractive index is the net value of the components of NE and indicates isotropic nature of formulation. The data in Table 3 indicates that the mean value of the refractive index for all the formulations was approxi-mately similar. All the drug free or drug loaded NE formulations had pH values ranging from 6.7 to 6.81, favorable for topical appli-cation [37]. It was observed that incorporation of drug did not sig-nificantly affect the pH values of NEs.

3.6. Thermodynamic Stability of Nanoemulsions

Stress test including heating cooling cycle, centrifugation and freeze thaw cycles showed that all the formulations had a good physical stability. After three months, domperidone was found to be stable with recovery > 97 % for all the formulations. No significant change in the mean values of the refractive index of the formula-tions was observed during 3 months (data not shown). Thus, it can be concluded that the NE formulations were not only physically stable but also chemically stable.

3.7. Ex-vivo Permeation Studies

The permeation ability of the selected NE formulations was evaluated by the ex-vivo permeation experiments. The permeation parameters of the tested NE and control formulations are presented in Table 4. The results demonstrate that the permeation rate and permeation coefficient of all the NE formulations through rat skin are significantly higher (P < 0.01) in comparison to control formu-lations D and E. The values of transdermal flux for different NE formulations were observed between 89.48 to 169.32 μg cm

-2 h

-1.

This value is approximately ten fold greater than the formulation D (15.75 ±05.90 μg cm

-2 h

-1), nearly twenty-seven fold higher than the

Table 3. Physical Characteristics of Nanoemulsion Formulations (Mean ± SD, n = 3)

Droplet Size (nm) Polydispersity Viscosity (mP) Refractive Index

Formulation

Placebo Drug Loaded Placebo Drug Loaded Placebo Drug Loaded Placebo Drug Loaded

NE-A1 38.02±1.46 57.03±3.28 0.167 ±0.06 0.172±0.05 64.00±3.40 62.89±3.09 1.401± .007 1.403±0.008

NE-A2 42.30±3.30 63.45±3.49 0.138± 0.09 0.137±0.02 79.40±3.65 79.99±3.33 1.403±0.009 1.400±0.009

NE-A3 47.02±3.70 70.50±3.89 0. 171±0.03 0. 179±0.09 97.30±2.89 98.07±3.19 1.406±0.009 1.399±0.005

NE-B1 32.00±2.88 48.09±2.75 0.125±0.05 0.128 ±0.04 28.55±2.03 28.70±2.82 1.400±0.005 1.402±0.007

NE-B2 34.00±2.86 50.64±2.53 0.165±0.04 0.155 ±0.10 43.30±3.09 43.89±2.51 1.409±0.008 1.405±0.006

NE-B3 36.10±1.97 54.00±3.12 0.159±0.07 0.158±0.08 52.70±2.90 51.81±3.03 1.405±0.006 1.401±0.003

NE-C1 47.40±2.90 71.48±2.83 0.172±0.09 0.179±0.11 100.00±3.40 103.57±3.38 1.402±0.004 1.409±0.007

NE-C2 51.10±1.98 76.50±3.33 0.161±0.05 0.171±0.07 131.10±5.40 132.09±4.37 1.400±0.003 1.403±0.005

NE-C3 58.55±3.50 87.82±4.04 0.178±0.07 0.181±0.09 155.34±6.30 155.97±4.95 1.406±0.008 1.398±0.002


formulation E (6.22 ±03.61 μg cm-2

h-1

) and fourteen fold higher than that reported for transdermal delivery of domperidone by Cal-pena et al. [28]. Although, all the formulations contain equal drug amount, low permeation rate as observed for formulations D and E, provided that concentration gradient is not a single factor affecting the rate of permeation.

Enhanced permeation with NEs could be explained on basis of the several mechanisms:

1) Nano droplets settle down to close contact with the skin and a large amount of inner oleic acid carrying drug might penetrate into the skin.

Fig. (3). The correlation between the concentration of polysorbate 20 in nanoemulsion formulations and the droplet size and viscosity of nanoemulsions (mean

value±S.D.; n = 3).

Table 4. Steady State Flux (Jss), Permeation Coefficient (Kp) and Enhancement Ratio (ER) of Domperidone Through Rat Skin from NE Systems

(NE-A1 to NE-C3), oil/Smix Mixture (D) and Oleic Acid (E) (mean ± SD, n = 3)

Formulationa

Jss

(μg h-1

cm-2

)

Kp

( x 103 cm h

-1)

ER

NE-A1 133.87 ±10.12c,d 0.022 ±0.0062 21.52

NE-A2 120.32 ±09.83c,d 0.020 ±0.0081 19.34

NE-A3 112.98 ±10.91c,d 0.018 ±0.0053 18.16

NE-B1 169.32 ±08.33c,d 0.028 ±0.0090 27.22

NE-B2 151.98 ±11.47c,d 0.025 ±0.0072 24.43

NE-B3 145.87 ±09.39c,d 0.024 ±0.0041 23.35

NE-C1 109.74 ±07.98c,d 0.018 ±0.0067 17.68

NE-C2 98.32 ±08.02c,d 0.016 ±0.0034 15.80

NE-C3 89.48 ±07.53c,d 0.014 ±0.0023 14.38

Db 15.75 ±05.90e 0.002 ±0.0004 2.53

E 06.22 ±03.61 0.001 ±0.0003 -

a All formulation carry equal drug load (domperidone, 6.0mg/mL) b Mixture of 1:9 ratio oleic acid and Smix (1:1 ratio of tween 20: carbitol) c P < 0.01 compared with D, d P < 0.01

compared with E, e P < 0.05 compared with E

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2) Permeation of drug carrying nano-sized droplets through stra-tum corneum without NE fusion and subsequent drug release.

3) Continuous and spontaneous fluctuating stable interfaces of NE enable high drug mobility and might enhance the drug dif-fusion process.

4) High solubilization of domperidone in NE resulting in high thermodynamic activity of the drug providing significant dri-ving force for its release and permeation.

5) Hydration of the stratum corneum due to external water phase of the NE results in high diffusivity of lipophilic drug as dro-plet size approaches to molecular dispersion.

In addition to this, significant enhancement (P<0.05) in flux was obtained for formulation D (15.75 ±05.90 μg cm

-2 h

-1) com-

pared to formulation E (6.22 ±03.61μg cm-2

h-1

). This was attrib-uted to the presence of Smix (polysorbate 20 and diethylene glycol monoethyl ether) in formulation D, which acted as efficient per-meation enhancers [39]. It was also observed that the flux of dom-peridone increases with the decrease in Smix ratio (Table 4) and the overall flux enhancements were observed for NE-B formulations with the maximum flux (169.32 ±08.33) for NE-B1, at 10 %

polysorbate 20. On plotting a graph between polysorbate 20 con-centration in the NE and corresponding flux values, a linear relation (r

2 = 0.9675) was obtained with decreased flux at increasing

polysorbate 20 concentrations (Fig. 4). This agrees well with results of Sintov et al., who also demonstrated high flux at low surfactant concentration [4]. In summary the different formulations can be ordered according to their descending flux values as follow: NE-B1 > NE-B2 > NE-B3 > NE-A1 > NE-A2 > NE-A3 > NE-C1 > NE-C2 > NE-C3 > formulation D > formulation E. Significantly low lag time was observed for all NE formulations ranging from 1.0 to 1.5 hr as compared to formulation D (6.0 ± 0.5 h) and E (11.1 ± 1.0 h). The reduced lag time as observed for NE formulations is useful for the anti-emetic therapy. Furthermore, for each NE formulation, curve estimation was also performed. The results of statistical analysis of curve estimation were used in order to develop regres-sion models that had the best r

2. Overall curve fitting showed that

the permeation profiles of NE followed the zero-order model (r2:

0.9972). Comparison of the permeation of the NE formulations with corresponding droplet size and viscosity indicated that NE-B1 with smallest droplet size and lowest viscosity had highest transdermal flux and therefore selected for in vivo permeation study.

Fig. (4). The correlation between the concentration of polysorbate 20 in nanoemulsion formulations and the flux of nanoemulsion formulations (mean

value±S.D.; n = 3).

Fig. (5). Pharmacokinetic profiles of domperidone following transdermal administration of drug loaded optimized nanoemulsion compared to orally adminis-tered drug suspension to eight rats (mean value±S.D.; n = 8).

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3.8. In vivo Permeation Studies

The AUC0 of topically applied NE-B1 formulation was 511.11 ± 31.8 ng/ml/h, while AUC0 of orally administered drug suspension was 144.80 ± 20.2 ng/ml/h. This resulted into 3.5 fold increased relative bioavailability (Fig. 5). The peak plasma concen-tration (Cmax) of domperidone after oral administration was found to be 45.0 ± 3.3 ng/ml at 0.5 ±0.1 h, while after topical administration of NE-B1 formulation it was 30.7 ± 2.1 ng/ml at 4.0 ± 0.5 h (Fig. 4). Low Cmax and prolonged tmax, might be due to the barrier proper-ties of the skin and slow partitioning of the drug into the skin from NE. The effective drug concentration (> 18 ng/mL in plasma) was maintained for at least 16 h through transdermal administration (Fig. 5).

The in vivo data, which have demonstrated significantly higher bioavailability of domperidone after transdermal administration of NE was due to avoidance of substantial amount of hepatic first pass metabolism associated with oral administration. The higher MRT values of transdermal delivery (10.96 ± 1.1 h) versus the oral route (2.84 ± 0.4 h) might be due to continuous replenishment of drug into the systemic circulation by controlled release of drug from the NE (Fig. 5). Thus transdermal administration of domperidone through oleic acid/ polysorbate 20/ diethylene glycol monoethyl ether/ water (o/w) NE had a sustained and enhanced absorption.

3.9. Skin Irritancy Test

The skin irritation test was performed to confirm the safety of the NE-B1 formulation. No erythema was observed over the period of six days and the components of the NE formulation were found safe for transdermal delivery.

4. CONCLUSION

The domperidone loaded thermodynamically stable o/w NE system were prepared and various formulation factors were evalu-ated to find optimized formulation which shows desirable efficacy both ex-vivo and in vivo. The optimized formulation NE-B1, which contained oleic acid (4 % w/w), polysorbate 20 (10 % w/w), dieth-ylene glycol monoethyl ether (20 % w/w) and water (64 % w/w) showed significant increase (P < 0.01) in the steady state flux (Jss) and permeability coefficient (Kp) compared to control or drug-loaded neat components. The in vivo studies revealed a 3.5 fold increased relative bioavailability. From ex-vivo and in vivo data it can be concluded that the developed nanoemulsion systems are potential vehicles for transdermal delivery of domperidone for pro-longed periods.

ACKNOWLEDGEMENT

We would like to thank Mr. Ravi Shankar Prasad, CDRI, Lucknow and Mr. Shadab A Pathan for their suggestions during pharmacokinetic studies.

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Received: July 09, 2008 Revised: August 05, 2008 Accepted: August 15, 2008

Investigation of Nanoemulsion System for Transdermal Delivery of Domperidone: Ex-vivo and in vivo...

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