PREPARATION AND CHARACTERIZATON OF ACECLOFENAC LOADED TOPICAL EMULGEL

ABOUT AUTHORS:
Sharma Devendra L.*¹, Dr. A.K.Seth¹, Nirmal Shah¹, Sachinkumar P Chauhan, Chintan Aundhia
¹Department of Pharmacy, Sumandeep Vidyapeeth University,
At & Po Pipariya, Ta.- Waghodia, Dist. Vadodara-391760.
(Gujarat) India
*9009dev@gmail.com

ABSTRACT
The main purpose of this work was to develop a topical emulgel formulation of Aceclofenac. Aceclofenac drug loaded formulation with Tween 80 as surfactant and PEG 400 as co-surfactant was developed  for topical application. Phase behavior and solubilization capacity of microemulsion system were characterized. Pseudo ternary phase diagram were developed by taking different concentration of Tween 80 and PEG 400 and the surfactant mixture containing tween 80 and PEG 400 ratio of (4:1) shows maximum amount of water. With the increase of tween 80 concentration, microemulsion region area and the amount of water and oil solubilsed into the microemulsion system increases, however increase of PEG 400 concentration produce opposite effects. The micromulsion system was also investigated in terms of other characterstics such as particle size, zeta potential, dilution test, centrifugation, conductivity, pH measurement, viscosity, % assay and % transmittance. Aceclofenac, poorly water soluble drug displayed high solubility in a microemulsion formulation using Capmul MCM oil (2.5%), Surfactant mixture (45%) and water (52.5%). The prepared micromulsion formulation was converted into gel form using carbopol 934 (1.5%) known as aceclofenac loaded topical Emulgel. The  ex-vivo study were performed and compared with marketed available gel.

REFERENCE ID: PHARMATUTOR-ART-1903

INTRODUCTION
Now a day, pharmaceutical research is aimed at developing suitable drug delivery system to fulfill the therapeutic needs of the patient. The development of novel drug delivery should meet with the development of new chemical entities, where majority of the molecules discovered were hydrophobic in nature. In the thrust of developing new delivery systems, Microemulsion (ME) gained a prominence in the effective delivery of the hydrophobic drugs. ⁽¹⁾
Aceclofenac [[[2-[(2, 6-Dichlorophenyl)- amino]-acetyl]-oxy]-acetic acid is a nonsteroidal anti-inflammatory drug (NSAID). It exhibits a multifactor mechanism of action which is mediated by selective inhibition of prostaglandin E2. The most widely cited side effect of NSAIDs includes, gastrointestinal ulcer, accompanied by anaemia due to the bleeding, which is also true for aceclofenac. In order to avoid the gastric irritation, minimize the systemic toxicity and achieve a better therapeutic effect, one  promising method is to administer the drug via skin.⁽²⁾
Transdermal drug delivery systems provide the most important way to achive these goals. The transdermal delivery system also enable controlled or sustained release of the active ingredients and an enhanced patient compliance. In this research, formulating topical microemulsions and in vitro permeation studies for aceclofenac was considered.⁽¹⁾

The topical application of NSAIDs  has been widely explored in the treatment of several disorders (e.g. osteoarthritis) as an alternative route to overcome the adverse side effects associated with the oral and rectal routes of administration, including gastrointestinal intolerance.  Several reports have shown that microemulsions represent promising vehicles for improving the delivery, efficacy and bioavailability of several NSAIDs, such as ketoprofen, celecoxib, rofecoxib, aceclofenac, piroxicam, and diclofenac. Microemulsions are thermodynamically stable, transparent, isotropic, low-viscosity colloidal dispersions consisting of oil and water stabilized by an interfacial film consisting of surfactant/cosurfactant. Microemulsions offer various advantages like thermodynamic stability, ability to entrap hydrophilic, hydrophobic therapeutic agents etc. These systems show the improved oral bioavailability due to enhanced solubility and permeability of the drug in the entire area of the gastro-intestinal tract.  Enhanced dermal transport was observed with these deliveries in the transdermal drug delivery due to lipophilicity and improved permeation rate in presence of surfactants. Besides these obvious advantages, also involves less number of unit operations in the formulation development, which is more convenient for the manufacturers in terms of economy. In addition microemulsion induces change in the thermodynamic activity of drug they contain, modifying their partition co-efficient and thus favours penetration through stratum corneum. Furthermore its components (Surfactant & Cosurfactant) reduce the functional barrier of the stratum corneum. ⁽²⁾

MATERIALS AND METHODOLOGY
Materials :
Aceclofenac was gifted by Modi pharmaceutical (Ahmedabad) , Capmul  MCM – National chemical (Baroda), Tween 80 – National chemicals (Baroda), PEG 400 – Suvidhinath laboratories (Baroda) and Carbopol 934 – Suvidhinath laboratories (Baroda). All other chemicals used were of analytical grade.

Screening of oils, surfactants and cosurfactants for microemulsion formation:
The most important criterion for the screening of oil for microemulsion is the maximum solubility and compatibility with drug. Basing on the biocompatibility profile of oils from literature review, drug solubility was determined. Excess amount of drug (100mg) in 3mL of selected oils (Iso-propyl myristate, Castor oil, Sesame oil, olive oil, Capmul MCM, Captax)was taken in stopper vials and was then mixed by vortex mixer. The mixture vials were then kept at 37±2.0 ^o C in an isothermal orbital shaker for 72 hr to reach equilibrium. The equilibrated samples were removed from shaker and centrifuged at 5000 rpm for 15 min. The solubility profile of drug in oil was determined from the supernatant using UV-VIS spectrophotometer at 278.2nm. Insoluble drug from the settled material was determined and mass balance was then found out. ⁽³⁾

Pseudoternary phase diagram :
Pseudo ternary phase diagram is a useful and important tool to study the  phase behaviour of microemulsions. Pseudo ternary phase diagram is constructed to obtain the appropriate components and their concentration ranges that can result in large existence area of microemulsion. Once the appropriate microemulsion components are selected, ternary pseudo phase diagram is constructed to define the extent and nature of the microemulsion regions. To produce such diagrams, a large number of samples of different composition are prepared. The microemulsion region is initially delineated by its isotropic nature and low viscosity. In this study, the Pseudo ternary phase diagrams of oil (Capmul MCM), surfactant/co surfactant (Tween 80/ PEG 400) and water were developed by using water titration method to obtain the components concentration ranges that can result in large existence area of microemulsion. Surfactant was blended with cosurfactant in fixed weight ratios (1:1, 2:1, 1:1, 3:1and 4:1). For each phase diagram, the ratio of oil to the Smix was varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 (w/w). Aliquots of each surfactant and cosurfactant mixture (Smix) were mixed with oil at room temperature with continuous stirring. Water was added drop wise to each oil-Smix mixture under vigorous stirring. After equilibrium, the samples were visually checked and determined as being clear microemulsions or emulsions or gels. Results are shown in Table 2 Phase diagram was plotted based on readings which are shown in Figure 1. ⁽¹⁾

Formulation of Microemulsion:
From the phase diagrams, Aceclofenac-loaded microemulsions were selected at different component ratios. Microemulsion systems were prepared by mixing oil with the mixture of surfactant and co-surfactant and water was added precisely drop by drop into oily phases with magnetic stirring at 37±0.5ºC (Table 2) The systems were equilibrated with gently magnetic stirring for 30 min followed by dissolving of appropriate amount of Acelofenac under ultrasonication.The final concentration of Acelofenac in microemulsion formulation was  2% w/w. ⁽⁴⁾

Preparation of Aceclofenac loaded topical emulgel:
Different concentration of Carbopol 934P (0.5%, 1% & 1.5%) and HPMC K4 M (0.5%, 1% & 1.5%)   were tried to prepare gel. Among them Carbopol 934P 1.5 %  gel was optimized for further process. In the small quantity of water, 1.5 % Carbopol 934P & 4.5% Propylene glycol (As Humectant) were mixed with stirring and allowed hydration for 4-5 hrs for gelling. ME was added in gel phase and left over night for gelling. Finally triethanolamine was added to adjust pH 6. ⁽¹⁾

CHARACTERIZATION OF MICROEMULSION:

Viscosity :
The viscositiy was measured to determine rheological properties of formulations. Brookfield Rheometer viscometer at 30?C with a CPE 61 spindle at 30 rpm was used to serve this purpose. Results were taken in triplicate and the average was taken in to consideration. ⁽⁵⁾

pH :
Another important parameter of microemulsion is pH. The excipients used in the formulation decide the pH of the final preparation and hence the route of administration. The change in the pH may affect the zeta potential of the formulation which in turn can affect the stability of preparation. The pH of the formulations was measured using digital pH meter. Results were taken in triplicate and the average was taken in to consideration.⁽⁶⁾

Drug content :
The drug content of Drug microemulsion formulation was measured using UV visible spectroscopic method. The 2 μg/ml of aliquot was prepared using microemulsion formulation using diluting solvent. The samples were measured as 278.2 nm using UV-VIS spectroscopic method. Results were taken in triplicate and the average was taken in to consideration. ⁽⁶⁾

Centrifugation :
This parameter was characterized to check the physical stability. The microemulsion system was centrifuged at 5000 rpm for 10 minutes to determine whether the system shows signs of creaming or phase separation. The system was observed visually for appearance. ⁽⁷⁾

Conductivity :
Electrical conductivity of formulated samples was measured using a digital conductometer at ambient temperature.Results were taken in triplicate and the average was taken in to consideration.⁽⁷⁾

Dilution test :
If the continuous phase is added in microemulsion, it will not crack or separate into phases. Maximum amount of water and oil were added to o/w and w/o formulations respectively and then inspected visually for clarity and phase separation. Here 50 and 100 times aqueous dilution of the formulation were visually checked for phase separation and clarity. Results were taken in triplicate and the average was taken in to consideration. ⁽⁸⁾

% Transmittance measurement :
Microemulsion formulation was diluted 50 and 100 times with distilled water. The percent transmittance of various formulations was measured at 630 nm using UV-VIS spectrophotometer against distilled water as a blank. Results were taken in triplicate and the average was taken in to consideration.⁽⁹⁾

Globule sizeand Zeta potential analysis :
Microemulsion formulation was diluted 50 times and 100 tomes with distilled water. The resultant samples were prepared by gentle agitation for 5 min using a magnetic stirrer. In addition, globule size distribution (PSD) and zeta potential of the final microemulsion were determined using dynamic light scattering technique by Malvern zetasizer (NANO ZS). Results were taken in triplicate and the average was taken in to consideration. ⁽¹⁰⁾

CHARACTERIZATION OF DRUG LOADED TOPICAL EMULGEL:

Viscosity :
The viscosity of the  formulation was measured to determine rheological properties of formulations. Brookfield LVDV II+ CP viscometer at 30?C with a CPE 64 spindle at 30 rpm was used to serve this purpose. Results were taken in triplicate and the average was taken in to consideration. ⁽¹¹⁾

pH :
The pH of the formulations was measured using digital pH meter. ⁽⁶⁾

% Drug Content :
The drug content of Drug topical microemulsion  formulation was measured using UV visible spectroscopic method. The 2 μg/ml of aliquot was prepared using formulation using diluting solvent. The samples were measured as 278.2 nm by UV-VIS spectroscopic method. Results were taken in triplicate and the average was taken in to consideration. ⁽¹¹⁾

Ex- Vivo Permeation Studies :
Ex-vivo drug permeation study of microemulsion formulations were carried out in Orchid Franz diffusion cell. Excised skin mucosa (ciliary portion facing towards donor compartment) was mounted on to the receptor compartment of the cell already containing 15 ml of phosphate buffer saline (PBS), pH 7.4 as permeation medium. Temperature was maintained at 37 ±2 ⁰C and rpm was set 25. Then on to that donor compartment was mounted. After a pre-incubation time of 10 minutes, pure drug solution (PDS) and developed formulations equivalent to 20 mg of Drug was put in the donor chamber. At predetermined time points, samples were withdrawn from the acceptor compartment, replacing the sampled volume with PBS pH 7.4 after each sampling, for a period of 12 hours. The samples withdrawn were first filtered, adequately diluted and were analysed as amount of permeated drug by UV-VIS spectrophotometer at 278.2 nm. Results were taken in triplicate and the average was taken in to consideration. ⁽⁶⁾

Stability of Drug microemulsion :
Samples of Drug microemulsion formulations and drug loaded emulgel were sealed in ampoules and then placed in Stability chambers at different temperature conditions i.e., room temperature (25±5?c, 60±5% RH) and accelerated condition (40±5⁰c, 75±5% RH) for 2 months. Duplicate samples were withdrawn at 0, 1 and 2 months to evaluate their physical and chemical stabilities. The physical stability was evaluated by visual inspection for physical changes such as phase separation and drug precipitation. Chemical stability was expressed as the content of Drug determined by UV visible spectroscopic method at 278.2 nm. ⁽¹²⁾

RESULT AND DISCUSSION

Result:

Solubilities study:- Result of solubility study of drug in oil , surfactant and cosurfactant are shown in Table 1.

Aceclofenac solubility study data :

Excipients	Solubility (mg/ml)
Oil
Iso propyl myristate	2.88
Capmul MCM	17.32
Olive oil	2.88
Castor oil	5.54
Captax	4.16
Surfactant
Tween 20	15.09
Tween 80	18.75
Co-surfactant
Propylene Glycol	5.20
PEG 400	28.64

Ternary phase diagram: surfactants: cosurfactants (4:1) show maximum microemulsion area. So this ratio was selected for further formulation. Phase diagram and exsitence  readings for microemulsion are shown in figure1 and Table 2.

Table 2  Smix and Micremulsion existence zone

Smix and Microemulsion existence zone
Figure No. 5.8	Surfactant: cosurfactant ratio	Microemulsion existence zone
A	1:2	+
B	1:1	++
C	2:1	+++
D	3:1	++++
E	4:1	+++++

( + shows range of microemulsion region )

Formulation of microemulsion:-

Table 3: Optimization of drug loaded microemulsion

Batch optimization
Batch No.	Batch Size (ml)	Oil (ml)	Smix (ml)	Water (ml)	% Transmittance	Centrifugation
ACF1	4ml	0.1ml	1.2ml	2.7ml	98.4%	X
ACF2	4ml	0.1ml	1.4ml	2.5ml	97.9%	X
ACF3	4ml	0.1ml	1.8ml	2.1ml	98.5%	X
ACF4	4ml	0.2ml	1.2ml	2.7ml	71.2%	X
ACF5	4ml	0.2ml	1.6ml	2.2ml	44.8%	X

Table 4: Composition of final microemulsion

S.no	Ingredient	% w/w
1	Capmul MCM	2.5
2	Smix( Tween 80:PEG 400) (4:1)	45
3	Water	52.5

Table 5: Composition of Emulgel

S.no	Ingredient	% w/w
1	Microemulsion	94
2	Carbopol Gel	1.5
3	Propylene Glycol	4.5

Characterization of Microemulsion:

Table 6: Characterization of Microemulsion

Characterization of Microemulsion
S.no	Test	Optimized drug loaded microemulsion
1	% Assay	91.0±0.2 %
2	pH	6.0±0.1
3	Conductivity	39±0.4%
4	% Transmittance	99.1±0.2%
5	Zeta potential(mV)	-2.06
6	Globule size (nm)	66.76
7	Weight/ml of ME	1.03±0.2 gm/ml
8	Viscosity (cp)	191.0
9	Zeta potential	-2.06mv
10	Particle size	65.76nm
11	PDI	0.663

Characterization of Microemulsion Gel:-

Table 7: Characterization of Gel

Characterization of Emulgel
S.No	Test	Result
1	% Assay	86.9±0.3 %
2	Transparency	Transparent & Clear
3	pH	6.5±0.2
4	Viscosity	1955.6 cp

The release profile of Aceclofenac:

Table 8: Ex-vivo drug release study

Ex-vivo drug permeation study
% Cumulative drug permeation
S.No	Time(hr)	Microemulsion (%)	Emulgel (%)	Marketed Gel (%)
1	1	07.96±0.7	04.81±0.4	04.42±0.3
2	2	13.20±0.4	09.25±0.2	08.18±0.7
3	3	18.00±0.8	12.33±0.6	11.19±0.5
4	4	24.53±0.9	16.52±0.6	14.25±0.9
5	5	29.87±0.4	21.78±0.3	18.74±0.6
6	6	35.42±0.6	26.23±0.8	23.05±0.6
7	7	41.56±0.8	32.45±0.4	28.96±0.3
8	8	45.44±0.3	36.41±0.8	32.81±0.8
9	9	51.62±0.6	40.90±0.5	37.76±0.9
10	12	62.78±0.6	54.12±0.7	48.65±1.0

Figure 2: Release profile

Stability study: Optimize preparation were checked Table 9, Table 10, Table 11 and Table 12 for stability study for 2 months. Results shown in table

Table 9: Result of stability study of microemulsion at room temperature

Stability study of microemulsion at room temperature
Test	Time Period
	Initial	1 Month	2 Month
Transmittance	99.1±0.1 %	99.0±0.2 %	99.0±0.2 %
% Assay	91.0±0.2 %	91.0±0.2 %	91.0±0.1 %
pH	6.0±0.1	6.0±0.1	6.0±0.2

Table 10: Result of stability study of emulgel at room temperature

 

Stability study of emulgel at room temperature
Test	Time Period
	Initial	1 Month	2 Month
% Assay	86.9 ±0.2 %	86.9±0.2 %	86.8±0.1 %
pH	6.5±0.1	6.5±0.1	6.5±0.1

 

Table 11: Result of stability study of microemulsion at accelerated temperature

 

Stability study of microemulsion at accelerated temperature
Test	Time Period
	Initial	1 Month	2 Month
Transmittance	99.1±0.1 %	99.0±0.2 %	99.0±0.2 %
% Assay	91.0±0.2 %	91.0±0.3 %	91.0±0.2 %
pH	6.0±0.1	6.0±0.2	6.0±0.1

 

Table 12 : Result of stability study of emulgel at accelerated temperature

Stability study of emulgel at accelerated temperature
Test	Time Period
	Initial	1 Month	2 Month
% Assay	86.9 ±0.1%	86.8±0.1 %	86.8±0.2 %
pH	6.5±0.2	6.5±0.1	6.5±0.2

 

Discussion:-
Solubility studies and selection of component:
From solubility study it was found that the solubility of the drug was higher in Capmul MCM oil. So Capmul MCM was selected as internal phase for the preparation of microemulsion. Tween 80 & PEG 400 were selected as surfactant & cosurfactant respectively based on solubility study.

Construction of phase diagram:
A Pseudoternary phase diagram of the investigated quaternary system water/Capmul MCM/Tween 80/PEG 400 was prepared by water titration method. Formation of microemulsion systems (turbid area) was observed at room temperature. Phase behavior investigations of this system demonstrated the suitable approach to determining the water phase, oil phase, surfactant concentration and co-surfactant concentration with which the transparent, one phase low-viscous microemulsion system was formed. The phase study revealed that the microemulsion region was found maximum when the surfactant-to-co-surfactant ratio was 4:1, so this ratio was taken for further study.

Optimization of microemulsion and emulgel:
Optimization of microemulsion formulation was done by choosing fixed quantity of oil phase and varying surfactant cosurfactant ratio. Here 5 drug loaded of different concentration of oil phase and surafactant cosurfactant mixture are prepared and among these based on stability and transparency optimize batch was selected.

Different concentration of carbopol 934 gel of 0.5%,1.0% and 1.5%  were prepared for ME gel (emulgel) . Among them carbopol 934 of 1.5% gel was optimized based on its viscosity , transparency and its consistency of application on skin.

Characterization of microemulsion and emulgel:
Qualitative test:-
From the dilution test and dye solubility test the prepared microemulsion was found to be o/w type.

Conductivity, % transmittance , pH and viscosity:-
As the system was showing good conductance , it was proved that the system was o/w type microemulsion. The developed microemulsion shows nearly 99% transmittance which proved good transparency of the system. The pH of the emulgel was found to be 6.5 which is suitable for application on skin and would not cause any irritation on skin. Viscosity was found to be satisfactory for both microemulsion and emulgel for topical application.

Globule size and Zeta potential:-
Globule size of the microemulsions was found to be 66.76 nm. The nanometric size range of the particle was retained even after 100 times dilution with water which proved the system’s compatibility with excess water. Zeta potential was found to be negative charge to the system --2.06. Hence the formulations will not cause any problem due to electrostatic interaction  between the microemulsion and skin on topical administration.

% drug release profile of Aceclofenac:-
In drug release profile, % Aceclofenac released from skin was found more in Microemulsion (62.78%) compared to Microemulsion gel (54.12%) and Market Product - Hifenac gel (48.65%). which showed drug release from Microemulsion is greater than emulgel and Market product, but the emulgel has better consistency for topical drug delivery, for that reason emulgel was selected for topical drug delivery system.

Stability study:-
Based on visual identification, microemulsion with Aceclofenac remained as clear liquid for a period of two months without the occurrence of phase separation or flocculation at the room temperature and accelerated  temperature as per ICH guidelines. The results of various studies performed on ME & emulgel were found to be satisfactory so both were found to be stable for period of two months.

CONCLUSION:
The present study concluded that Capmul MCM based Aceclofenac loaded emulgel drug delivery system can be  an effective novel topical formulation  for the fast relief of  chronic pain of joints due to arthritis.  However, further studies in animals and human being need to be performed before this formulation can be commercially exploited.

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