INVESTIGATION OF HYDROGEL THICKENED MICROEMULSION FOR TOPICAL ADMINISTRATION OF MELOXICAM

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ABOUT AUTHORS:
Tulsi. P. Upadhyay*, Jitendra Patel, Harsh. A. Vyas, Nirali. N. Thakkar, Kruti. C. Patel, Umesh. M. Upadhyay
Department of Pharmaceutics,
Sigma Institute of Pharmacy,
Vadodara, Gujarat, India
*tulsiupadhyay90@gmail.com

ABSTRACT:
The present study was conducted to investigate the microemulsion based gel of meloxicam in order to bypass gastrointestinal side effects with more patient compliance. Microemulsion existence range was defined by pseudo ternary phase diagram. Pseudoternary phase diagrams were developed for combination of Iso propyl Myristate (oily phase), Tween80:PEG400 (surfactant : cosurfactant) and water (aqueous phase) with Aqueous phase titration method. Various microemulsion formulations were prepared and further evaluated for various parameters like pH, conductance, transmittance, drug release, etc.Optimized formulation of Microemulsion was thickened with gelling agent Carbopol 940 to yield a gel with desirable properties facilitating the topical application. Safety of formulation was evaluated using skin irritancy test. Simple Meloxicam gel and optimized microemulsion gel then subjected to in vitro drug release comparison study. Drug exhibited maximum solubility in Iso propyl Myristate as oily phase among all selected oils and maximum solubility in Tween80 and Polyethylene Glycol 400(PEG400) as surfactant and cosurfactant. The pseudoternary phase diagram has been delineated at surfactant : cosurfactant ratio 2:1. Microemulsion showed -0.5 zeta potential which is desirable for its stability and average particle size was obtained less than 200nm. Microemulsion based gel afford better drug release when compared to simple gel. Present work concluded that microemulsion based gel can be promising formulation for application of Meloxicam with good patient compliance too in treatment of rheumatoid arthritis, osteo arthritis and ankylosing spondylitis.

REFERENCE ID: PHARMATUTOR-ART-2206

INTRODUCTION:
1.1 INTRODUCTION TO TRANSDERMAL DRUG DELIVERY SYSTEM
Transdermal drug delivery systems are dosage forms designed to deliver a therapeutically effective amount of drug across a patient’s skin. In order to deliver therapeutic agents through the human skin, the comprehensive morphological, biophysical and physicochemical properties of the skin are to be considered. Transdermal delivery provides a leading edge over injectable and oral routes by increasing patient compliance. So transdermal drug delivery - an approach used to deliver drugs through the skin for therapeutic use as an alternative to oral, intravascular, subcutaneous and transmucosal routes. The human skin is a readily accessible surface for drug delivery^1,2. Subcutaneous layer is the principle barrier layer of skin. Any topical dosage form has to cross it to penetrate inside the skin.

The microemulsion concept was introduced as early as the 1940s by Hoar and Schulman³ who generated a clear single-phase solution by titrating a milky emulsion with hexanol. Subsequently the term microemulsion defined and indeed redefined on many occasions. Thus it defined as “a system of water, oil and amphiphile which is a clear and thermodynamically stable liquid solution.”

There are three types of microemulsions which are most likely to be formed depending on composition^4,5. O/W type, W/O type and Bicontinuous.

The miscibility of oil, water and amphiphile (surfactant plus co-surfactant) depends on the overall composition, which is system specific. Ternary (water/amphiphile/oil) and quaternary (water/surfactant/co-surfactant/oil) phase diagrams can describe the phase manifestations and are essential in the study of microemulsion. Phase diagram is the vital step to know the existence of microemulsion. Such characteristics textures are commonly referred as Winsor phases^5,6and the classification is distinguished as below.
· Winsor I: with two phases, the lower or single phase region of common micelles (o/w) microemulsion phase in equilibrium with the upper excess oil;
· Winsor II: with two phases, the upper microemulsion phase or a reverse micelle phase (w/o) in equilibrium with lower excess water;
· Winsor III: with three phases, middle microemulsion phase so called bicontinuous phase (o/w plus w/o) in equilibrium with upper excess oil and lower excess water;
· Winsor IV: in single phase, with oil, water and surfactant homogeneously mixed.

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Much attention has been focused on the topical delivery of many drugs through microemulsions. Microemulsions are well known to improve the absorption and bioavailability of many compounds so their topical application is widespread, especially in the case of anti-inflammatory drugs or hormones. Skin is very big barrier in transportation of drug. So from microemulsion lipophilic domain of emulsion interact with stratum corneum so increase permeability while hydrophilic domain hydrate stratum corneum and this will increase lamellar volume of lipid bilayer and thus interfacial structure disrupt and so facilitation of drug transport through skin.

Meloxicam is a potent non-steroidal anti-inflammatory (NSAID) drug^7,8 used orally to alleviate the symptoms of osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis.NSAID inhibits cyclooxygenase (COX), the enzyme responsible for converting arachidonic acid into prostaglandin H₂-which are mediators of inflammation. Meloxicam is enolic acid class of oxicam derivatives which shows preferential inhibition of cyclo-oxygenase-2 (COX-2) over COX-1.It is believed that local accumulation of the drug intarget tissues could occur either by direct penetration or via redistribution through systemic circulation. It has been suggested that drugs passing through the stratum corneum, epidermis, and dermis can be effectively removed by cutaneous microcirculation, which can act as a “sink”(Yue Y et al,2012). Meloxicam is practically insoluble in water and soluble at higher pH. As being NSAID it is having gastric side effects. So to overcome problems like side effects and water solubility microemulsion based gal can be the best dosage form. It is locally acting dosage form so will give fast relief in symptoms and also better patient compliance due to less side effects. Aim of present study was to overcome drug side effects and to enhance patient compliance with suitable dosage form.

MATERIALS AND METHODS
2.1. MATERIALS
Meloxicam was gifted by Apex Healthcare, Gujarat, India. IPM, PEG 400, Triethanolamine and Tween 80 were obtained from Qualikem Fine Chemicals Pvt. Ltd., New Delhi, India. Carbopol 940 was obtained from Loba chem., Vadodara, India.

2.2. Screening of oils, surfactants and co- surfactants for microemulsion^2,9:
The solubility¹⁰ of meloxicam in various oils (IPM, IPP, oleic acid, Eucalyptus Oil, Castor oil), surfactants (Tween 20 & tween 80), cosurfactants (PG and PEG 400) was determined by dissolving an excess amount of meloxicam in 2 mL of each of the selected oils, surfactants, and co-surfactants in 5 mL capacity stoppered vials separately to determination of solubility. They were stirred continuously for 72 h at 37 ± 1°C. After equilibrium was attained, the mixture was centrifuged at 3000 rpm for 10 min; the supernatant layer was carefully removed and then diluted with a solution. The concentration of drug was then measured using UV/Visible spectrophotometer by comparison with a standard calibration curve.

2.3Construction of Pseudo-Ternary phase Diagrams¹¹:
· Phase Titration method:
In order to find out the concentration range of components for the existence range of microemulsions, pseudo-ternary phase diagrams were constructed using H₂O titration method at ambient temperature (25 ºC). Three phase diagrams were prepared with the weight ratios 1:1, 2:1and 3:1 of Tween 80 to PEG 400 respectively. For each phase diagram at specific surfactant/co-surfactant weight ratio, the ratios of IPM to the mixture of surfactant and co-surfactant were varied as 1:9 to 9:1. These mixtures were titrated drop wise with water under gentle magnetic stirring. After being equilibrated the systems were visually characterized. Persistent turbidity up to 24hr considered as end point. Transparent fluid systems were characterized as microemulsion. Highly viscous systems that did not show a change in the meniscus after being tilted to an angle of 90^º were considered as gel.

2.4 Method of preparation of meloxicam loaded microemulsion:
Meloxicam was added to the oil. Varying ratios of S_mix (surfactant + cosurfactant) and water was taken. Meloxicam microemulsion was obtained by adding oil and drug mixture to water phase drop by drop with moderate stirring at ambient temperature.

2.5 Method of preparation of microemulsion based gel^12,13:
Gelling agents were evaluated for their ability to Gel meloxicam microemulsion. Briefly, meloxicam microemulsion was prepared as per section 2.4. Then gelling efficiency of gelling agents like poloxamer 407(20%)^14,15 and carbopol 940(1%)¹⁶were decided. After optimization plain gel of desired gelling agent i.e Carbopol 940 was prepared with 3 different concentrations to get better gel with optimum concentration.

After formation of plain gel prepared microemulsion was added to that gel upto its efficiency. Efficiency of gel for having microemulsion with desired characteristics was checked. Then amount of Meloxicam for 2gm gel was calculated. 0.4%w/w¹⁷gel was prepared.

2.6 Method of Preparation of Simple Meloxicam Gel¹⁸:
Meloxicam gel formulation was prepared by using Carbopol 940 as gelling agent. Plain carbopol gel with concentration of 2.5% was prepared with water. Meloxicam was dissolved in PEG400 and then poured into previously prepared plain gel. 0.4% Meloxicam gel was prepared and then compared with results of microemulsion gel.

CHARACTERIZATION:
3.1 CHARACTERIZATION OF MICROEMULSION:
· Particle size, Size distribution and Zeta potential:
The particle size, size distribution and zeta potential were determined using a computerized inspection system with zetasizer.

· Determination of pH¹⁹:
The pH values of 1% aqueous solutions of the prepared microemulsions were measured by a pH meter. The glass electrode was calibrated with two standard buffers (pH of 4.00 and 9.00). The preparation was left for about 15 min for attaining equilibrium while measuring.

· Viscosity:
Viscosity of microemulsion formulation was measured by Brookfield Viscometer. Apparent viscosity was measured rotating the RV spindle 2 at 2 and 5 rpm at 30ºC.

· % drug Content:
1 ml microemulsion dissolved in solvent mixture (Ethanol + 0.1N NaOH) and measure drug content through UV spectrophotometer at 364 nm²⁰

· Percent transmittance (%T)²¹:
Transparency of microemulsion formulation was determined by measuring the percentage transmittance with purified water taken as blank through UV spectrophotometer.

· Conductance²²:
The conductive measurements were taken by a conductivity meter. The microemulsion prepared with addition of water was measured after thorough mixing and temperature equilibration at 25°C, the electrode was dipped in the microemulsion sample until equilibrium was reached, and reading becomes stable.

· In-vitro drug release study^23,24:
An essential parameter in the evaluation of drug delivery is the rate at which the drug is released from the carrier. Skin permeation study with drug-containing microemulsion formulation was carried out using modified Franz diffusion cell. Full thickness abdominal skin of male Wister albino rats weighing 140 to 200 g was used for the skin permeation. Briefly, to obtain skin, animal was sacrificed. Hair from the abdominal region was carefully removed and an excision in the skin was made. The dermal side of the skin was thoroughly cleaned of any adhering tissues. Dermis part of the skin was wiped 3 to 4 times with a wet cotton swab soaked in isopropanol to remove any adhering fat. The skin specimen was cut into appropriate size after carefully removing subcutaneous fat and washing with normal saline.

Skin was mounted in a modified Franz diffusion cell, kept at 32±0.5ºC. The known quantity of microemulsion equivalent to 8 mg of drug was put uniformly on the skin on donor side. PH 7.4 phosphate buffer¹⁷was used as the acceptor medium, from which samples were collected at regular intervals. Collected samples were estimated with UV spectroscopy at 364nm. All permeation experiments were repeated three times and data were expressed as mean of three experiments ± standard deviation (SD).

3.2 CHARACTERIZATION OF MICROEMULSIONGEL:

· Determination of drug content:
For determination of drug content, about 1 g of the gel is been weighed in a 100-ml volumetric flask and dissolved in suitable solvent. It is diluted appropriately and drug content is been determined at 364 nm using Shimadzu – 1700 UV Visible spectrophotometer.

· pH:
The pH value of 1% aqueous solutions of the prepared gels is measured by a pH meter. The glass electrode is calibrated with two standard buffers (pH of 4.00 and 9.00).The preparation is left for about 15 min for attaining equilibrium while measuring.

· Viscosity:
Microemulsion based gel is evaluated for viscosity under constant temperature, RV spindle 2 and 5 rpm by using Brookfield viscometer at 30ºC.

· Drug Release:
An essential parameter in the evaluation of drug delivery is the rate at which the drug is released from the carrier. Skin permeation study with drug-containing microemulsion based gel formulation was carried out using modified Franz diffusion cell. Full thickness abdominal skin of male Wister albino rats weighing 140 to 200 g was used for the skin permeation. Briefly, to obtain skin, animal was sacrificed. Hair from the abdominal region was carefully removed and an excision in the skin was made. The dermal side of the skin was thoroughly cleaned of any adhering tissues. Dermis part of the skin was wiped 3 to 4 times with a wet cotton swab soaked in isopropanol to remove any adhering fat. The skin specimen was cut into appropriate size after carefully removing subcutaneous fat and washing with normal saline.

Skin was mounted in a modified Franz diffusion cell, kept at 32±0.5ºC. The known quantity of microemulsion based gel equivalent to 4 mg of drug was put uniformly on the skin on donor side. pH 7.4 phosphate buffer was used as the acceptor medium, from which samples were collected at regular intervals. Collected samples were estimated with UV spectroscopy at 364 nm. All permeation experiments were repeated three times and data were expressed as mean of three experiments ± standard deviation (SD).

· Calculation of steady state skin flux (J _flux) of formulation²⁵:
Skin flux (mg/cm²/h) (J_flux) is determined from Fick’s first law of diffusion:
J_flux= (dM/dt) × V / A

Where,
dM/dt = amount of meloxicam penetrated per unit time
A = effective diffusion area
V= volume of receiver compartment (ml).

Steady state skin flux (J_flux) was determined from the slope (dM/dt) of the linear portion of a cumulative penetration time curve.

· Kinetic modelling^6,19:
In order to understand the kinetics and mechanism of drug release, the results of in vitro drug release are fitted into various kinetic equations like zero order (cumulative % release vs. time), first order (log % drug remaining vs. time), Higuchi’s model (cumulative % drug release vs. square root of time), Krosmeyer peppas plot (log of cumulative % drug release vs. log time). R² (coefficient of correlation) were calculated for the linear curve obtained by regression analysis of the in vitro drug permeation plots.

· Primary skin irritation studies²⁶:
The optimized gel formulation was evaluated for skin irritation studies on healthy rats. The hairs of the dorsal portion were removed physically with the help of sharp surgical scissors and the skin was washed properly one day prior to use. Rats were divided into four groups (six rats in each group):
Group I: Normal: No treatment was given.
Group II: Blank: Blank gel (without drug) was secured.
Group III: Medicated: Medicated gel (with meloxicam) was secured.
Group IV: Formalin: 0.8%v/v aq. solution of Formalin was applied as a standard irritant

The gel was removed after each 48 hr. and the area examined for any signs of skin sensitivity or irritation and the fresh gel was secured at the same site at 2nd, 4th, 6th day. All the respective treatments were continued till 7 days and finally application sites were monitored visually.

RESULTS:

4.1 Solubility:
4.1.1 Solubility studies of meloxicam in oils:

Table 1: Solubility studies of meloxicam in oils

OILS	SOLUBILITY(mg/ml)
Iso propyl myristate(IPM)	2.88±0.05
Iso propyl palmitate(IPP)	1.85±0.04
Oleic acid(OA)	1.33±0.02
Castor oil	1.17±0.03
Eucalyptus oil	0.98±0.07

N=3

Figure 1: Solubility studies of meloxicam in oils

4.1.2 Solubility studies of meloxicam in surfactants:

Table 2: Solubility studies of meloxicam in surfactants

SURFACTANTS	SOLUBILITY(mg/ml)
TWEEN80	5.674±0.04
TWEEN20	4.973±0.04

N=3

Figure 2: Solubility studies of meloxicam in surfactants

4.1.3 Solubility studies of meloxicam in co-surfactants:

Table 3: Solubility studies of meloxicam in co-surfactants

COSURFACTANTS	SOLUBILITY(mg/ml)
PEG 400	10.213±0.1
PG	8.909±0.06

N=3

Figure 3: Solubility studies of meloxicam in co-surfactant

4.2 RESULTS OF PSEUDO TERNARY PHASE DIAGRAM:

Figure 4: Pseudo ternary phase diagrams (a) 1:1, (b) 2:1, (c) 3:1 ratio of S_mix i.e. s:co::Tween80:PEG 400

Table 4: Formulations of microemulsions of 2:1 ratio of Surfactant: co-surfactant

BATCH	IPM (%)	TWEEN 80: PEG400 (%) (2:1)	WATER (%)
F1	4	35	61
F2	5	40	55
F3	5	45	50
F4	6	60	34
F5	7	43	50
F6	9	55	36
F7	9	45	46
F8	9	35	56
F9	10	45	46
F10	10	55	36

Table 5: Evaluation of microemulsion formulation

BATCH	pH	%DRUG CONTENT	%TRANSMITTANCE
F1	6.10±0.01	69.25±0.04	81.3±0.14
F2	6.56±0.01	74.34±0.05	85.7±0.14
F3	6.12±0.00	75.23±0.02	82.3±0.14
F4	7.12±0.01	79.17±0.03	83.9±0.00
F5	7.01±0.02	85.16±0.03	92.3±0.14
F6	6.71±0.01	97.99±0.04	99.8±0.07
F7	6.39±0.01	92.64±0.04	99.6±0.14
F8	6.52±0.01	90.94±0.02	94.2±0.14
F9	6.22±0.01	84.33±0.03	89.7±0.14
F10	6.28±0.02	85.51±0.04	99.6±0.07

N=3

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Table 6: Evaluation of microemulsion formulation

BATCH	CONDUCTANCE (μs/cm)	%CUMULATIVE DRUG RELEASE IN 240min.	VISCOSITY (Spindle 5)
			2 RPM	5 RPM
F1	145.5±0.85	47.45±0.55	61±1.41	56±2.45
F2	170.2±1.13	59.87±0.29	54 ±5.66	50±2.56
F3	168.3±0.99	73.49±0.71	48±3.54	45±0.12
F4	165±2.33	91.54±1.13	72±2.12	68±4.67
F5	147.4±2.47	91.84±0.35	58±2.83	53±1.32
F6	165.6±1.27	95.91±0.64	67±0.71	59±2.45
F7	140.4±1.56	93.34±1.27	62±1.41	56±1.78
F8	132.3±0.64	74.89±0.55	78±0.00	71±1.41
F9	173.7±1.48	79.41±0.04	81±1.41	73±5.66
F10	178.2±1.7	81.21±1.32	75±4.24	63±2.83

N=3

Figure 7: Zeta potential of Microemulsion Batch F6

Figure 8: Size Distribution of Microemulsion Batch F6

4.3. Formulation Table of Microemulsion Gel:

Table 7: Formulation table microemulsion based gel of optimized microemulsion batch F6

INGREDIENTS	G1	G2	G3
MELOXICAM (%)	0.4	0.4	0.4
IPM (%)	9	9	9
TWEEN80:PEG400 (%)	55	55	55
Water (%)	36	36	36
Carbopol 940	1.5	2	2.5
Triethanolamine	q.s	q.s	q.s

4.4. Results of Evaluation of microemulsion based gel:

Table 8: Evaluation of Microemulsion Based Gel

Sr. No	Viscosity(Cps) (2 RPM)	Drug Content (%)	Drug Release (%)
1	4500±124.72	94.89±1.13	76.10±0.12
2	5200±124.72	95.34±0.8	78.28±0.9
3	6100±169.97	97.75±0.65	100±0.1

N=3

Figure 9: Cumulative Drug Release of batches G1-G3

PharmaTutor (ISSN: 2347 - 7881) Volume 2, Issue 7 Received On: 15/05/2014; Accepted On: 18/05/2014; Published On: 01/07/2014 How to cite this article: TP Upadhyay, J Patel, HA Vyas, NN Thakkar, KC Patel, UM Upadhyay; Investigation of Hydrogel Thickened Microemulsion for Topical Administration of Meloxicam; PharmaTutor; 2014; 2(7); 120-134