Nanoemulsion based gel for transdermal delivery of meloxicam: Physico-chemical, mechanistic investigation

Life Sciences 92 (2013) 383–392

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Nanoemulsion based gel for transdermal delivery of meloxicam: Physico-chemical, mechanistic investigation S. Khurana a, N.K. Jain b, P.M.S. Bedi a,⁎ a b

Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar – 143104, India Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar – 470003, India

a r t i c l e

i n f o

Article history: Received 5 September 2012 Accepted 7 January 2013 Keywords: Nanoemulsion gel Meloxicam Transdermal delivery In vitro skin permeation Skin interactions Carrageenan induced paw edema model

a b s t r a c t Aims: The aim of the present investigation was to develop a nanoemulsion (NE) gel formulation for the transdermal delivery of meloxicam (MLX) in order to ensure maximum controlled and sustained drug release capacity. Main methods: The MLX containing NE gel was prepared and characterized for particle size, zeta potential, pH, rheology, in vitro drug release, in vitro skin permeation, and in vitro hemolysis. Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) of MLX-NE gel treated rat skin was performed to investigate the skin permeation mechanism of meloxicam from NE gel. Skin permeation potential of the developed gel formulation was assessed using confocal laser scanning microscopy (CLSM). The in vivo toxicity of MLX-NE gel was assessed by histopathological examination in rat. The rat paw edema test was performed to evaluate the anti-inflammatory activity of MLX-NE gel. Key findings: Percutaneous absorption studies demonstrated a higher permeation of meloxicam from NE gel, than the drug solution. FTIR and DSC studies supported stratum corneum lipid extraction as a possible penetration enhancer mechanism for MLX-NE gel. CLSM studies confirmed the permeation of the NE gel formulation to the deeper layers of the skin (up to 130 μm). MLX-NE gel turned out to be non-irritant, biocompatible, and provided maximum inhibition of paw edema in rats over 24 h in contrast to MLX solution. Significance: The nanoemulsion gel formulation may hold promise as an effective alternative for the transdermal delivery of meloxicam. © 2013 Elsevier Inc. All rights reserved.

Introduction Meloxicam (MLX) is a non-steroidal anti-inflammatory drug used orally to relieve the symptoms of osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis (Eglitis et al., 1996; Burke et al., 2006). It is emerging as a promising drug for the treatment of Alzheimer's disease and as a viable adjuvant therapeutic agent for the treatment of various types of cancers (Wolfesberger et al., 2006; Goldman et al., 1998). Although, meloxicam preferentially inhibits COX-2 (cyclooxgenease-2) over COX-1 (cyclooxgenease-1), in practice it still has incidence of gastrointestinal side effects at high doses on long term treatment (Villegas et al., 2001). It was noted in the UK Drug and Therapeutics Bulletin that “there is no convincing evidence that the risk of the severest adverse gastrointestinal events, namely peptic ulceration, perforation and bleeding, is lower with meloxicam than with other NSAIDs” (Distel et al., 1996; Lanes et al., 2000). Therefore, there is a need of a delivery system for meloxicam, which modulates gastric side effect and delivers the drug to the inflammatory site. Although, transdermal administration of ⁎ Corresponding author. Tel./fax: +91 183 2258819. E-mail address: [email protected] (P.M.S. Bedi). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.01.005

NSAID offers the advantage of delivering a drug directly to the disease site in order to maximize local effects without concurrent systemic activity yet, no formulation of meloxicam is available in the market for transdermal use. The most difficult aspect of the transdermal delivery system is the formidable barrier properties of intact skin that prevent percutaneous absorption of drugs. Although meloxicam possesses some favorable properties for transdermal administration like low molecular weight, low daily therapeutic dose yet the inherent poor aqueous solubility and high melting point make them unsuitable for transdermal delivery. It does not exhibit enough lipophilicity for permeation across the skin. A number of transdermal drug delivery systems, which vary in their compositions and structures such as gels (Jantharaprapap and Stagni, 2007; Ruiz Martinez et al., 2007; Yuan et al., 2007a,b; Kasliwal et al., 2008), microemulsions (Yuan et al., 2006), and transdermal patch (Ah et al., 2010), have been developed to improve the skin permeation of meloxicam. However, the poor drug loading capacity, poor drug controlled and sustained release capacities have limited their use as transdermal carriers. The level of interest in nanoemulsions (NE) as carrier systems in transdermal drug delivery has increased substantially. They are optically transparent nanometricsized emulsions with particle sizes between 100 and 500 nm, composed

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of the oil, surfactant, co-surfactant and water (Gutierrez et al., 2008; Kong and Park, 2011). However, the application of the nanoemulsion to the skin is inconvenient due to low viscosity (Lawrence and Rees, 2000). To increase their viscosity and to make them more suitable for the transdermal application, gelling agents can be used (Klang et al., 2012). Caprylic acid (CA) based nanoemulsion as the transdermal drug carrier was developed in our previous study (Khurana et al., 2010), which was converted into gel form and physicochemically characterized in this article. To validate the transdermal use of the MLX-NE gel, its interaction with the skin was studied. Ex vivo skin permeation and skin deposition, in vivo anti-inflammatory activity, and in vivo toxicity were studied.

Instruments, UK). The morphology of gel was examined by Transmission Electron Microscopy (TEM) (Morgagni 268D, FEI, Holland). The pH measurements of NE gel were determined using a calibrated digital pH meter (Hanna instruments HI 9321, Michigan, USA) in investigating samples. Drug entrapment efficiency (E.E) was calculated by determining the amount of drug entrapped (A2) after removal of unentrapped drug using a dialysis bag (Sigma, USA; MWCO 12,000–14,000) (Washington, 1989; Khurana et al., 2012) by using Eq. (1): E:Eð%Þ ¼ ðA2 =A1 Þ  100

ð1Þ

Materials and methods

where A1 is the total amount of drug added in the formulation. The concentration of drug was determined by UV spectrophotometry (Hitachi U-2800 spectrophotometer, Tokyo, Japan) at 362 nm.

Materials

Rheology

Meloxicam was obtained as gift sample from M/s Lupin Pharmaceuticals Ltd., Goa, India. Caprylic acid, propylene glycol (PG), Tween 80, polyethylene glycol 400 (PEG 400), and triethanolamine were purchased from S. D. Fine Chemicals, Mumbai, India. Carrageenan, Carbopol 940, Trypsin (Type III bovine), Rhodamine 123, HPLC grade of methanol, water and phosphoric acid were purchased from Sigma-Aldrich, Mumbai, India. Dialysis membrane (MWCO 12-14,000) was purchased from Hi-Media, Mumbai, India. All other chemicals were of the analytical grade and used as received.

The flow behavior of the NE gel formulations was studied in a Brookfield Viscometer (Model RVT, Brookfield Engineering Laboratories, Inc., USA) at rotational speed of 0.5, 1.0, 2.0, 2.5, 4, 5, 10, 20, 50, and 100 rpm using spindle ≠ 2. The position of the upward and downward curves in the rheogram was studied at 25 ± 1 °C to determine the flow behavior of the different formulations.

Methods Preparation of nanoemulsion gel (NE gel) The optimized NE was prepared according to the procedure previously described by our group (Khurana et al., 2010). An appropriate amount of distilled water was added to the heated (65 °C) caprylic acid under magnetic stirring to obtain homogeneous milky slurry to which a calculated volume of Tween 80 and the propylene glycol stock solution were added. The mixture turned transparent within seconds due to the formation of the nanoemulsion. For the preparation of nanoemulsion gel (plain-NE gel), Carbopol 940 was added to the formed nanoemulsion with constant stirring. Triethanolamine was then added under gentle stirring and the pH was adjusted to 6.0. The gel was allowed to stand overnight to remove entrapped air. Meloxicam and Rhodamine 123 containing nanoemulsion gel (MLXNE gel) were prepared after replacing a definite amount of the lipid phase by MLX and Rhodamine 123, respectively and processed in the same way as mentioned above. Solutions containing meloxicam and Rhodamine 123 were prepared by dissolving meloxicam and Rhodamine 123, respectively in water containing Tween 80 and PG (Table 1). Physico-chemical characterization The particle size analysis of the prepared gel formulation (diluted with distilled water) was performed by photon correlation spectroscopy (PCS) with a Malvern Nanosizer ZS (Malvern Instruments, UK). Zeta potential (ZP) was measured in a Malvern Nanosizer ZS (Malvern

Spreadability (www.floratech.com) To assess the spreading properties of the gel formulation, the weighed cellulose acetate filter paper (W1) was placed in the center of the aluminum foil sheet. Several milliliters of the tested formulation were filled into the 5 ml syringe (BD syringe, Becton Dickinson & Co. USA). Then, 20 drops of the formulation were pushed out of the syringe over the defined area in the center of the cellulose acetate filter paper. After 10 min, saturated portion of the filter paper was cut away from the unsaturated portion. The unsaturated portion of the filter paper was weighed out accurately (W2) and % Spread by weight was calculated using the Eq. (2) %Spread by Weight ¼ ½ðW1 –W2 Þ=W1   100:

ð2Þ

In vitro release The in vitro release of MLX from MLX-NE gel was evaluated using a dialysis membrane (MWCO 12–14,000). The membrane was mounted between the donor and receptor compartments of a locally fabricated Franz diffusion cells (diffusion area of 2.26 cm 2; receptor volume of 22.5 ml). Because of the low solubility of MLX in water and in buffer solution, acetate buffer of pH 6.0 with 30% PEG 400 (v/v) was selected as receptor medium and maintained at 32±0.5 °C in the receptor compartment. Unentrapped drug was removed from the formulation by ultra dialysis against acetate buffer (pH 6.0) with 30% PEG 400 at 4 °C. Two hundred and fifty milligram of the dialysate was then applied evenly on the surface of the membrane in the donor compartment. The aliquots from the receptor compartment were withdrawn at predetermined time

Table 1 Composition of investigated formulations. Formulation

Meloxicam (mg)

Rhodamine 123 (mg)

Caprylic acid (mg)

Tween 80 (ml)

Propylene glycol (ml)

Carbopol 940 (mg)

Water (ml)

Plain-NE gel MLX-NE gel Rhodamine123-NE gel MLX solution Rhodamine 123 solution

– 5

– – 5 – 5

100 95 95 – –

0.2 0.2 0.2 0.2 0.2

0.1 0.1 0.1 0.1 0.1

5 5 5 – –

0.7 0.7 0.7 0.7 0.7

5

Plain-NE gel, nanoemulsion gel; MLX-NE gel, nanoemulsion gel loaded with meloxicam; Rhodamine 123-NE gel, nanoemulsion gel loaded with Rhodamine 123; MLX solution, meloxicam solution.

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interval and replaced immediately with a similar volume of fresh medium. The samples were centrifuged, and analyzed for drug content by HPLC (high-performance liquid chromatography) assay. The experiment was performed in triplicate. Additionally, the MLX solution (5 mg/ml) acted as a control. The data obtained from in vitro release studies was fitted to different kinetic models (Zero order, First order and Higuchi) to find out the mechanism of meloxicam release from NE gel. Ex vivo skin permeation and skin penetration studies Ex vivo skin permeation and skin penetration studies were performed in a Franz diffusion cell with a diffusion area of 2.26 cm2 and receptor volume of 22.5 ml using abdominal rat skin. Abdominal rat skin was excised and washed with isotonic NaCl. The excised skin was then mounted between the donor and receptor chambers of the Franz diffusion cell with the dermal side in contact with the receptor medium and the stratum corneum side facing upwards into the donor compartment. Then, 250 mg of the formulation was evenly applied on the surface of the rat skin in the donor compartment. The receptor compartment was filled with acetate buffer (pH 6.0) containing 30% PEG 400 and stirred continuously. The temperature in the receptor compartment was maintained at 32±0.5 °C to simulate the skin temperature. At predetermined time intervals (1, 2, 3, 5, 7, 9, 12, and 24 h), 1.5 ml samples were collected from the receptor compartment and replaced with a fresh receptor solution to maintain sink condition. All the collected samples were centrifuged and analyzed for meloxicam content by HPLC. The MLX solution (5 mg/ml) served as control. For evaluation of drug penetration into rat skin, the formulation remaining on the skin surface was removed by gentle washing with PBS (pH 7.4) at the end of the experiment (24 h). The stratum corneum (SC) was removed by stripping the skin surface with hypoallergic, transparent adhesive tape (Transpore 3 M surgical tape, 3 M India Ltd, India). The epidermis was separated from the dermis with a surgical sterile scalpel (Alves et al., 2007). To extract the drug, tape strips, epidermis, and dermis were placed each in methanol and sonicated for 20 min. All the samples were then centrifuged, the supernatants collected were analyzed for drug content by HPLC. Permeation data analysis Meloxicam steady state flux (Jss, μg/cm 2/h) (permeation rate) across the skin was calculated by dividing the slope of the linear portion of the curve of cumulative amounts of the drug permeated and time by the area of the skin surface through which diffusion took place. Permeability coefficient (Kp) was calculated by using the following Eq. (3) Kp ¼ Jss  Co

ð3Þ

where, Co is the initial drug concentration in the donor compartment. Enhancement ratio (Er) was calculated by dividing Jss of the respective formulation by Jss of the control formulation (Ustundag Okur et al., 2011). High-performance liquid chromatography (HPLC) estimation of meloxicam The equipment consisted of a Varian Prostar 210 HPLC system equipped with an autosampler Varian 410 and a diode array detector Varian 330 (Varian Deutschland GmbH, Darmstadt, Germany). The chromatographic separation was performed at room temperature using a spherisorb 5 μm RP-C8 column (250 mm×4.6 mm, Supelco, Milano, Italy) with a flow rate of 1.0 ml/min. Twenty microliters of sample was injected into the column. The mobile phase consisting of methanol, water and phosphoric acid (69.9:30:0.1) was delivered at a flow rate of 1 ml/min. The detection wavelength was 362 nm. The elution period

385

was 8 min while the retention time of was about 5.8 min. All operations were carried out at ambient temperature. MLX-NE gel skin interaction study Differential scanning calorimeter (DSC, Mettler Toledo, 821e, Switzerland) and FTIR spectrophotometer (Perkin Elmer, Germany), were used to study the effect of the MLX-NE gel on excised rat skin (Fang et al., 2008). The excised rat skin samples were cleaned with isotonic NaCl solution and subcutaneous fatty tissue was removed. The SC was separated from a skin by digestion with 0.1% trypsin in a phosphate buffer solution (pH 7.2) at 37 ± 0.5 °C for 4 h. The isolated SC was rinsed with cold distilled water and dried by storage in desiccators over silica-gel (Azeem et al., 2012). The SC was cut into circular pieces with an approximate diameter of 2.3 cm. These samples of SC were incubated in a Franz diffusion chamber at 32 ± 0.5 °C with 250 mg of the MLX-NE gel formulation for 24 h. After incubation, the remnants of formulation sticking on the surface of treated SC samples were cleaned off. Untreated SC was used as a control. Delipidized SC was prepared by placing the untreated stratum corneum in chloroform/methanol mixtures, 2:1 (Swartzendruber et al., 1987) for 48 h at room temperature and was used as negative control. DSC thermogram (DSC, Mettler Toledo, 821e, Switzerland) and FTIR spectrum (FTIR spectrophotometer, Perkin Elmer, Germany) of skin SC were recorded. The DSC parameters were evaluated using STARe Software (Mettler Toledo, 821e, Switzerland). In vitro hemolysis assay In vitro hemolysis assay of the MLX-NE gel and its individual components (at the concentration used in the MLX-NE gel), was performed using citrated human blood freshly drawn from the antecubital vein (Kong and Park, 2011; Li et al., 2010). 10 ml blood sample was centrifuged at 3000 rpm for 10 min and the supernatant was discarded. The erythrocytes were washed with isotonic saline solution three times to remove debris and serum protein and were diluted with PBS (7.4) to a final concentration of 2% (v/v). Test sample (1 ml) was added to a 100 μl aliquot of the erythrocyte stock dispersion and incubated at 37 ± 0.5 °C for a period of 1 h. After incubation, the mixtures were centrifuged at 1500 g for 10 min to remove, debris and unlysed erythrocytes. The supernatants were analyzed for released oxyhemoglobin content spectrophotometrically at a wavelength of 546 nm (Hitachi U-2800 spectrophotometer, Tokyo, Japan). Triton X-100 solution (5% v/v), and PBS (pH 7.4) were taken as positive and negative control, respectively. The hemolysis rate was calculated from the following equation Hemolysis rate ð% Þ ¼ ðAs −A0 Þ=ðA100 −A0 Þ

ð4Þ

where As is the absorbance of the test sample, A0 and A100 are the absorbance of the negative control and the positive control, respectively. Animal studies Albino rats (180–200 g) of either sex were used for in vivo studies. The animals were housed in polypropylene cages under standard laboratory conditions and had free access to food and water ad libitum. The study protocol was approved by the Institutional Animal Ethical Committee of Guru Nanak Dev University, Amritsar, India. Visualization of skin penetration in vivo Albino rats (180–200 g) of either sex, weighing 150–200 g were randomly divided into two groups of three rats each. The skin of the dorsal region was trimmed free of hair by shaving. 5 mg/ml solution of marker Rhodamine 123 and NE gel formulation loaded with Rhodamine

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123 (5 mg/ml) were applied in a marked area of 1 cm2 at the dorsal site of animals of group I and group II, respectively for 24 h. Thereafter, animals were sacrificed by cervical dislocation. The excised skin was blotted in inert paper, washed thrice with ethanol. The application area was then sectioned into the pieces of 1 mm2 size and evaluated for depth of probe penetration using Confocal Laser Scanning Microscope (LSM510, Carl ZEISS, Germany) (Dubey et al., 2007). In vivo toxicities In vivo toxicity test was conducted in mice to evaluate the potential toxicity of the MLX-NE gel after transdermal application. Eighteen mice (20–25 g) were randomly divided into three equal groups. The first group (untreated) served as a control. The dorsal skin was trimmed free of hair by shaving. Plain NE-gel and MLX-NE gel at the dose of 15 mg/kg body weight were applied to the dorsal site of the animals of group II and group III, respectively. After 24 h of application of the formulation, the remnants of the gel were gently washed away from the skin surface using adsorbent cotton dipped in 0.9% w/v physiological saline. The dorsal site of the treated animal was then visually inspected for any erythema. Thereafter, animals were sacrificed by cervical dislocation. Liver, kidney, stomach, and heart were taken out. The excised skin and isolated organs were preserved in 10% formalin for histopathological examination. Sections were fixed and blocks were made using the procedure as reported (Chen-yu et al., 2012). The sections were stained with eosin–hematoxylin to determine gross histopathology. Histological sections were examined using phase contrast microscope with photographic arrangement (Zeiss, Primastar). Carrageenan paw edema test The anti-inflammatory activity of gel formulations was studied by carrageenan induced rat paw edema volume model. The rats (150–180 g) were randomly divided into four groups of six rats each. A mark was made at the ankle joint of each animal. MLX-NE gel, plain-NE gel, and MLX solution (250 mg each) were applied on the subplantar region of the left hind paw of first, second, and third groups, respectively. Fourth group was untreated and served as control. 1 h post transdermal application, paw edema was induced by subplantar injection of 0.1 ml of a 1% w/v freshly prepared carrageenan in normal saline into the left hind paw of each rat. The paw volume up to the ankle joint was measured before and at different time intervals after the carrageenan injection using graduated plethysmograph (INCO, India). Percentage reduction in edema was calculated using the following formula (Gupta et al., 2002).

% reduction in edema ¼

%edema ðcontrolÞ−%edema ðtreated groupÞ : %edema ðcontrolÞ

ð5Þ

The mean particle size of plain-NE gel and MLX-NE gel was found to be 123±1.5 nm and 125±1.9 nm, respectively with polydispersity indices (PI) below 0.2. The zeta potential of plain-NE gel and MLX-NE was found to be −30.66±0.51 mV, and −31.85±0.61 mV, respectively (Table 2). There were statistically insignificant differences (p>0.5) between plain and MLX loaded NE gel in terms of their particle size and zeta potential. The drug entrapment efficiency of the MLX-NE gel was found to be 89.61±1.45% (Table 2). TEM (Morgagni 268D, FEI, Holland) images revealed that the nanoemulsion droplets in Plain-NE gel and MLX-NE gel were non-aggregated and nearly spherical in shape (Fig. 1a,b). The pH of both plain and meloxicam loaded NE gels was found to be 6.0 (Table 2).

Rheology Rheology is an important parameter as it affects the spreadability and adherence of transdermal formulations to the skin surface. Gel formulations were stored at 25 ± 1 °C for 5 days before the investigation of their rheological behaviors. The rheogram of plain-NE gel and MLX-NE gel displayed Newtonian flow behavior as indicated by superimposed ascending and descending flow curves of the rheogram (Fig. 2). The flow curves were unaffected (p > 0.5) by the presence of MLX.

Spreadability From a patient compliance point of view, spreadability is a pivot for transdermal formulation (Chow et al., 2008). The semi-solid MLX-NE gel was found to exhibit good % spread by weight (57.42 ± 1.30) that would assure the practicability to skin administration (Table 2). There was insignificant difference (p> 0.5) between the spreadability of plain-NE gel and MLX-NE gel.

In vitro release The ability of gel formulation to deliver MLX was examined by determining the drug release rate. Fig. 3 shows the cumulative percentage release of meloxicam from MLX-NE gel and MLX solution at different sampling intervals. An increased drug release rate was achieved in MLX solution as compared to MLX-NE gel. MLX was released in a controlled manner from MLX-NE gel and 95.04 ± 0.19% of meloxicam was released within 24 h, in contrast to 92.05 ± 0.78% of meloxicam released from MLX solution within 7 h. In order to describe the drug release profiles from the MLX-NE gel and control formulation, the in vitro release data were fitted into Zero order (Qt vs. t), Higuchi (Qt vs. t 1/2), and First order kinetic model (log Qt vs t), where Qt is the cumulative percentage of drug released at time t. Drug release from both MLX-NE gel and MLX solution followed Zero order release kinetics with a best fit r 2 value of 0.994 and 0.991, respectively.

Statistical analysis All the results are expressed as mean±standard deviation (SD). Data was analyzed using student's t-test or one-way ANOVA followed by Tukey or Dunnett's t-test (Sigma Stat Software, 2.03); p-valuesb 0.05 were considered as statistically significant. Results Physicochemical characterization In this work, plain and meloxicam loaded nanoemulsion gel were prepared using caprylic acid as the oil phase, Tween 80 as the surfactant, PG as the co-surfactant, and Carbopol 940 as a gelling agent (Table 1).

Table 2 Characteristics of nanoemulsion gel (NE-gel). Parameter Particle size (nm) PI Zeta potential (mV) Drug entrapment efficiency (%) pH % Spread by weight Steady state flux (Jss, μg/cm2/h) Permeability coefficient (Kp, cm/h) The data reported are mean ± s.d. (n = 3).

Plain-NE gel

MLX-NE gel

123 ± 1.5 0.182 ± 0.01 −30.66 ± 0.51 – 6.0 57.83 ± 1.30

125 ± 1.9 0.193 ± 0.01 −31.85 ± 0.61 89.61 ± 1.45 6.0 57.42 ± 1.30 6.407 ± 0.0911 0.0057

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387

b

a

Fig. 1. Transmission electron micrographs (TEM) of (1a) nanoemulsion gel (plain-NE gel) and (1b) meloxicam loaded nanoemulsion (MLX-NE) gel.

Ex vivo skin permeation study The gel formulations were characterized ex vivo for skin permeation profiles and the results are shown in Fig. 4. The cumulative amount of drug permeated at the end of 24 h was found to be 344.061 ± 1.49 μg and 186.34 ± 1.66 μg for MLX-NE gel and MLX solution, respectively (Fig. 4). MLX-NE gel effectively enhanced meloxicam permeation compared to MLX solution, showing high ER (1.89), reflecting a high flux value (6.407 ±0.0911 μg cm2/h) with relatively high skin permeation rate constant (0.0057 cm/h) (Table 2). The penetration ability of MLX-NE gel to deliver the drug into the rat skin was investigated and results clearly demonstrated that the epidermal and dermal levels of meloxicam in case of MLX-NE gel at 24 h was 3.24, 1.42 times higher as compared to the MLX solution control, respectively. On the other hand, SC level of meloxicam in case of MLX solution at 24 h was 1.02 folds high as compared to the MLX-NE gel. MLX-NE gel skin interaction study FTIR analysis Fig. 5(A)–(C) shows the FTIR spectra of untreated (control), MLX-NE gel treated, and delipidized (negative control) skin SC, respectively over the wave number range 650–4000 cm −1. The spectrum of normal untreated rat skin SC showed the major absorption peaks at 2850.72 cm −1 and 2919.24 cm −1 due to symmetrical and asymmetrical C\H stretching vibrations of the lipid alkyl chains, respectively; the ester band at 1739.72 cm−1, a strong band at 1637.98 cm−1 due to C_O stretching vibrations of amide 1 band, and at 1544.26 cm−1 due to N\H bending vibrations of amide II (Fig. 5A). A decrease in the peak height and area of the C\H stretching bands

was observed in MLX-NE gel treated SC as compared to the control SC (Fig. 5B). On the other hand, these bands were absent in dilapidated SC (Fig. 5C). Other bands did not show any shift or change in band intensity in and MLX-NE gel treated SC (Fig. 5B) and delipidized SC (Fig. 5C). Differential scanning calorimetry Fig. 6(A)–(C) shows the DSC thermogram of the control, MLX-NE gel treated, and delipidized (negative control) rat skin SC, respectively. Untreated rat skin (control) SC showed three endothermic peaks at temperatures 65 °C (T1), 82 °C (T2) and 104 °C (T3) (Fig. 6A). A less commonly reported peak around 32–40 °C, attributed to superficial sebaceous lipids was not observed in our experiments, which might be attributed to the low enthalpy of this peak (Lee et al., 2005). In the case of skin samples pretreated with MLX-NE gel, the lipid transition at 65 °C (T1) and 82 °C (T2) disappeared and broad new transition at 80 °C appeared in the DSC thermogram (Fig. 6B). Lipid transition at 65 °C (T1) and 82 °C (T2) also disappeared from the DSC thermogram of negative control SC samples (Fig. 6C). In vitro hemolysis assay From the in vitro study, hemolysis of the erythrocytes to some extent by the excipients of NE gel was confirmed. Triton-X 100 (hemolytic agent) demonstrated 99.94% hemolysis of erythrocytes (Fig. 7). MLX-NE gel showed very slight hemolytic activity that had been greater than the caprylic acid yet lower than the hemolysis caused by the other excipients (P b 0.05). In accordance with the previous reports, this end result might be associated with the changes in the

70

Shear Stress (τ)

60

UpcurveMLX-NE gel DowncurveMLXNE gel Upcurve plainNE gel Downcurve plain-NE gel

50 40 30 20 10 0 0

20

40

60

80

100

120

Shear Rate (RPM) Fig. 2. Rheogram of nanoemulsion gel (plain-NE gel) and meloxicam loaded nanoemulsion (MLX-NE) gel.

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100

%Cumulative drug release

90 80 70

MLX-NE gel

60 MLX solution

50 40 30 20 10 0 0

5

10

15

20

25

30

Time (h) Fig. 3. In vitro release profile of meloxicam from meloxicam containing nanoemulsion gel (MLX-NE gel) and meloxicam (MLX) solution.

mode and degree of interactions between the components of the formulation and erythrocytes during processing of nanoemulsion gel. For lipid emulsions and nanostructured lipid carriers, comparable findings have already been reported (Jumaa et al., 1999; Nayak et al., 2010).

of inflammation like inflammatory infiltrate, and edema in the dermis. Compared to control, no histopathological changes were seen in the liver, kidney, stomach and heart of plain-NE gel and MLX-NE treated animals. These results indicated that developed nanoemulsion gel is safe for transdermal delivery of meloxicam.

Visualization of skin penetration in vivo

Carrageenan induced edema model

The depth of Rhodamine 123 loaded nanoemulsion gel penetration was measured by CLSM. As shown in Fig. 8a, NE-gel was effective in permeating Rhodamine 123 up to 130.0 μm, while Rhodamine solution was confined to 20.0 μm depth (Fig. 8b).

The anti-inflammatory and sustaining action of all the formulations was evaluated by the carrageenan induced hind paw edema method in rats. The mean percentage inhibition of edema in the MLX-NE gel, and MLX solution as determined by a one way ANOVA differed significantly with p b 0.05. The percent inhibition was found to be greater for MLX-NE gel (17.14% mean inhibition, after half an hour and 70.58% after 24 h) as compared to MLX solution which exhibited mean percentage edema inhibition of 20%, after an hour and 17.64% after 24 h (Fig. 10).

In vivo toxicity After application of MLX-NE gel and plain-NE gel on the dorsal site of hairless mice skin for 24 h, skin irritation and histopathological changes were investigated. No obvious skin erythema and edema were visible in animals of treated and untreated groups. The photomicrographs of skin histological sections of animals treated with MLX-NE gel, plain-NE-gel, and untreated animals are shown in Fig. 9a–c, respectively. H and E stained sections of control skin sample showed epidermis consisting of a cornified squamous layer and underlying germinative and granular layers. The dermis showed collagen fibers, small capillaries, hair follicles and sebaceous glands in subepidermal region. In the deeper region layer adipose cells and striated muscles were seen. No inflammatory infiltrate, granulomatous pathology or evidence of malignancy was seen (Fig. 9a). The MLX-NE gel (Fig. 9b) and plain-NE gel (Fig. 9c) did not show signs

Discussion The primary objective of this study was the development of NE gel utilizing different skin permeation enhancers to control the delivery of meloxicam into the skin upon transdermal application to ensure that high amount of the drug may be accomplished in inflammatory tissue without any toxicity. Saturated as well as unsaturated fatty acids have been extensively analyzed as penetration enhancers primarily for lipophilic substances (Fox et al., 2011). Here, we selected caprylic acid (eight-carbon saturated fatty acid) as the oil phase for the preparation of the nanoemulsion gel and tested the potential of

Cumulated drug permeated (μg)

400 350 300 250

MLX-NE gel

200 150 MLX solution 100 50 0 0

5

10

15

20

25

30

Time (h) Fig. 4. Ex vivo skin permeation profile of meloxicam from meloxicam loaded nanoemulsion gel (MLX-NE gel) and meloxicam (MLX) solution across the excised rat skin.

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Fig. 5. FTIR spectra of untreated (A), meloxicam loaded nanoemulsion gel (MLX-NE gel) treated (B), and delipidized (C) rat skin stratum corneum.

formulated nanoemulsion gel to promote transdermal delivery of meloxicam. First of all, we succeeded in developing negatively charged nanoemulsion gel containing caprylic acid as oil phase. The small mean particle size and PI values below 0.2 (Table 2), indicated a narrow droplet size distribution and thus assured better stability against destabilization phenomena such as Ostwald ripening (Klang et al., 2012). MLX-NE gel with a pH value (6.0) near to the normal skin showed Newtonian flow with good spreadability, which would assure the practicability to skin administration. In order to evaluate the skin targeting potential of the NE gel, the skin permeation and penetration ability of meloxicam across the rat skin were examined in vitro. Compared to MLX solution, the skin permeation and penetration ability were more pronounced with MLX-NE gel. MLX-NE gel

revealed continuous permeation of meloxicam across the skin and the quantity of meloxicam penetrated into the stratum corneum was lower than that observed in the epidermis and dermis. This is of interest for transdermal application of MLX-NE gel as sufficient drug levels could reach deeper skin layers which is the main target site for drug action where they can act as a drug reservoir. The enhanced skin permeation and penetration of meloxicam from MLX-NE gel may be attributed to its compositions in which the ingredients such as caprylic acid (short chain fatty acid), Tween 80, and PG that act as permeation enhancers and could significantly reduce the barrier properties of stratum corneum. On the contrary, Ki and Choi (2007) reported very low permeation rate of meloxicam from Tween 80, and propylene glycol solution across hairless mouse skin over 36 h. The difference in the results may be attributed to the fact that these components act synergistically and significantly reduced the barrier properties of stratum corneum (Williams and Barry, 2004). Moreover, the small droplet size of nanoemulsion provides a very large surface area for drug transfer into the skin. In addition, 120

%Hemolysis

100 80 60 40 20 0

Sample Fig. 6. DSC thermogram of untreated (A), meloxicam loaded nanoemulsion gel (MLX-NE gel) treated (B), and delipidized (C) rat skin stratum corneum.

Fig. 7. % hemolysis produced by meloxicam loaded nanoemulsion gel (MLX-NE gel), and excipients used in the production of nanoemulsion gel.

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Fig. 8. Confocal laser scanning micrograph of rat skin after (a) 24 h treatment with Rhodamine 123 containing nanoemulsion gel (Rhodamine 123-NE gel) and (b) 24 h treatment with Rhodamine 123 solution.

the nanoemulsions can interact with the lipid bilayers of the stratum corneum, and thereby contribute significantly to the penetration enhancing effect of NE gel (Karande and Mitragotri, 2009). In order to elucidate the possible changes caused by MLX-NE gel on the structure of the skin and, thus to elucidate their penetration enhancement mechanism, FTIR and DSC studies were undertaken out. It is of great potential for studying the lamellar lipid structure of SC that primarily provides barrier attributes to the SC. The SC predominantly consists of

corneocytes tightly packed with keratin that are interconnected by a multilamellar lipid bilayer structure composed predominately of ceramides, cholesterol, fatty acids, cholesterol esters, and cholesterol sulfate (Al-Saidan, 2004; Bentley et al., 1997). In our study, the FTIR spectrum of the control SC over the wave number range 650–4000 cm−1 provided absorption peaks at different wave numbers which correlate well with the values found in the literature (Hasanovic et al., 2011; Schwarz et al., 2012). These peaks are mainly related to lipid and protein

Fig. 9. Photomicrographs of skin sections of (a) untreated animals, (b) animals treated with meloxicam containing nanoemulsion gel (MLX-NE gel) and (c) nanoemulsion gel (plain-NE gel) treated.

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Mean percentage edema inhibition

80 MLX solution

70 60

MLX-NE gel 50 40 30 20 10 0 1

2

3

4

5

6

12

24

Time (h) Fig. 10. % reduction in edema produced by meloxicam containing nanoemulsion gel (MLX-NE gel), and meloxicam solution (MLX solution) in carrageenan induced rat paw.

molecular vibrations in the SC and provide information regarding the interior structure of the SC lipids (Suhonena et al., 1999). The characteristic stretching bands observed in control SC at 2850.72 cm−1 and 2919.24 cm−1 were reported to be associated with the lipid order in SC, and the complete absence of both symmetric and asymmetric C\H stretching bands in the negative control SC samples further proved the association of these bands with the intercellular lipids. The FTIR spectrum of MLX-NE gel treated SC demonstrated a prominent decrease in peak height and area of these symmetric and asymmetric C\H stretching bands. As height and area of these C\H stretching band are proportional to the amount of the lipids present (Laugel et al., 2005), the decrease in peak height and area of these bands in MLX-NE gel treated SC indicated the extraction of SC lipids. This observation is in agreement with that obtained by Azeem et al. (2012) and supports the theory that the MLX-NE induced permeation enhancement in transdermal transport of meloxicam might be attributed to the formation of the pores in the lipid bilayer, and therefore creating an increased effective volume inside SC lipid domain available for drug diffusion (Krishnaiah et al., 2002; Rastogi and Singh, 2001). Then, the impact of the formulation on the amide I and II bands to elucidate the change in protein conformation in the stratum corneum was analyzed. A blue shift in amide I and a red shift in amide II band frequency would signify the permeation enhancing effect of the applied formulation. However, no significant peak shift in the amide I and II bands could be observed in our study. To further elucidate the mechanism of skin permeation enhancement of the MLX-NE gel, its impact on the thermal properties of the SC intercellular lipids were studied by DSC. Results of DSC study demonstrated the greater effect of nanoemulsion gel on stratum corneum lipids responsible for T1 (65 °C) and T2 (82 °C) transitions. The complete absence of T1 and T2 lipid transition in the negative control skin SC samples proved the association of these transitions with the intercellular lipids of the skin (Zellmer et al., 1995; Carelli et al., 1998; Changez et al., 2006). This suggests that the MLX-NE gel can cause certain modifications in the lipid structures of SC and also that the SC lipids extraction is an important mechanism involved in enhancing its permeation ability. It is well known that the ability of a formulation to cause hemolysis of red blood cells is predictive of local tissue irritancy (Brown et al., 1989). Consequently, just before examining the in vivo tolerance of the MLX-NE gel, different ingredients of nanoemulsion gel and conventional drug solution were put through hemolysis assays and the results evinced the hemocompatibility of nanoemulsion gel. To assess the in vivo skin penetration ability of MLX-NE gel and to confirm the finding of in vitro skin permeation studies, CLSM of rat skin treated with NE gel containing Rhodamine 123 (lipophilic fluorescence marker) instead of drug was performed. The results of the study demonstrated that NE could penetrate the deeper

layers of rat skin (up to 130 μm), while Rhodamine solution remained confined to the SC (20 μm) only (Fig. 8a,b). These results are consistent with those of in vitro skin permeation study. It can be concluded that NE gel is more effective in improving the skin permeation of meloxicam and these characteristics are significant for its end use performance as transdermal dosage form. Some studies have demonstrated that meloxicam oral therapy has also been associated with gastric ulceration, renal damage, respiratory and/or urticaria/angioedema type hypersensitivity reactions, the risk of thomboembolic events (Singh et al., 2004). In vivo skin tolerance was assessed by histopathological examination of the MLX-NE gel treated and control rat skin, and the excellent skin tolerance of the developed MLX-NE gel was observed. In order to rule out the possibility of toxicity on internal organs, commonly associated with the oral use of Cox-2 selective anti-inflammatory drugs, histopathological examinations of stomach, kidney, heart and liver were performed after transdermal application of the MLX-NE gel on rats. No histopathological changes could be detected in the microscopic examination of these organs and biocompatibility and safety of the developed MLX-NE gel was thus confirmed. The carrageenan-induced edema test demonstrated that the MLX-NE gel possessed the strong anti-inflammatory activity as compared to drug solution. The enhanced anti-inflammatory effects of formulation MLX-NE gel could be ascribed to the high release rate and enhanced skin permeation of meloxicam across the skin. The drug release from MLX solution was not enough to control edema effectively for multiple hours. These results suggest the potential of NE based gel for transdermal application of MLX-NE gel without any side effects. Furthermore, they alleviate the various disadvantages like low drug loading capacity, less skin permeation ability, chemical instability, and high cost associated with other carriers like liposomes, niosomes, and nanoparticles.

Conclusion The results obtained in the present work show that NE gel containing caprylic acid as oil phase is a suitable carrier system for the incorporation of meloxicam, and satisfies the best attributes for transdermal application i.e., Newtonian flow, good spreadability, and suitable release profile. This debut study suggests that caprylic acid based nanoemulsion gel could reduce the SC barrier effects and could enhance the transdermal permeation and penetration of MLX. MLX-NE gel exhibited excellent skin tolerance, hemocompatibility and significant efficacy in inflammation when compared to MLX solution. Results are encouraging and substantiate the role of meloxicam containing NE gel as an effective anti-inflammatory therapy.

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Conflict of interest The authors report no declarations of interest.

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