Research Article

Investigation of potential of organogel carrying etodolac for anti-inflammatory activity

Shashikant Pawar1, Amol Jahagirdar1, Dnyaneshwar Kolkar1, Moreshwar Patil1*, Pavan Udavant2, Sanjay Kshirsagar1

1Department of Pharmaceutics, MET's Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik 422 003 (M.S), India

2Department of Pharmacology, MET's Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik 422 003 (M.S), India

 

*For correspondence

Dr. Moreshwar Patil,

Associate Professor, Department of Pharmaceutics, MET's Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik 423301, Maharashtra, India.

Email: moreshwarp_iop@bkc. met.edu

 

 

 

 

Received: 03 December 2015

Revised: 15 December 2015

Accepted: 21 December 2015

ABSTRACT

Objective: The aim of the present investigation was to formulate and evaluate stable etodolac organogel preparation which will increase the solubility of etodolac and releases the drug for prolonged time period.

Methods: The amount of the Span 80 and Tween 80 was added to the sesame oil containing propyl paraben. Etodolac was added to the mixtures of oil to formed gelator solution. Subsequently, water containing methyl paraben was added with constant stirring on magnetic stirrer until there was formation of organogel. Carbopol 934 was added to maintain the consistency of organogel. Formulated organogels were evaluated for their physical appearance, pH, viscosity, globule size, drug content, spreadability, gel-sol transition study, in vitro and ex vivo drug release, anti-inflammatory activity and stability.

Results: Formulated organogel showed good physical appearance, acceptable skin pH (6 - 6.8), non-newtonian pseudoplastic system, drug contents (96.36±1.75), globule size (337.3 nm), spreadability (6.45±0.055 gm.cm/sec), gel-sol transition (650C) for selected batch, good extrudability, in vitro release (96.36%), ex vivo release (73.10%), skin irritation study did not showed any irritation reaction and possess a good anti-inflammatory activity.

Conclusions: The drug release of selected batch OG3 showed 96.36% as compared to marketed gel. Similarly ex-vivo release of formulation showed 73.10% release through mice skin compared with marketed gel.

Keywords: Organogel, Anti-inflammatory activity, gel sol transition, globule size, ex-vivo release

Introduction

Inflammation is defined as the local response of living mammalian tissues to injury due to any agent. It is a body defense reaction in order to eliminate or limit the spread of injurious agent followed by removal of the necroses cells and tissues. "Immunity" or "Immune reaction" and inflammatory response by host are to be protective mechanism in the body, inflammation is the visible response to an immune reaction and activation of immune response is almost essential before inflammatory response appears.[5] When tissue injury occurs whether caused by bacteria, trauma chemicals, heat or any other phenomenon, multipherubtorces are released by injured tissues caused by dramatic secondary changes in surrounding uninjured tissue called inflammation.1

A gel may be defined as a semi-solid formulation having an external solvent phase, a polar (organogel) or polar (hydrogel) immobilized within the spaces available of a three dimensional networked structure.[15] Organogels are gels based on non-aqueous liquids, which have been mentioned in various Pharmacopoeias as useful topical deliveries for lipophilic drugs. Organogel not only exert a local effect but also are capable of achieving systemic effect through percutaneous absorption, when their lipophilic nature and occlusive effect are potentiated by the presence of a penetration enhances.18

Organogels preparations for external application to skin have gained much demand, because it is easily absorbed through the skin layers. In general, organogels are thermodynamically stable in nature and have been explored as matrices for the delivery of bioactive agents. In the last decade, interest in physical organogels has grown rapidly with the discovery and synthesis of a very large number of diverse molecules, which can gel organic solvent at low concentrations.19

In a recent development Span in conjugation with the some other additive have been shown to provide a very promising topical drug delivery vehicle i.e. organogel. The formation of three-dimensional in the organogel is the result of transition at micelles level in low viscous network liquid consisting of span cause micelles in non-polar organic liquid. These spherical reverse micellar states of lipid aggregates, Tween on to form elongated tubular micelles with the addition of after and subsequently entangle to form a temporal three dimensional network in the solution bulk.18

Organogels have been studied to have many applications in pharmaceuticals, nutraceuticals, cosmetics, food and so on. The scope of organogels further increases, since; the topical route becomes one of the convenient methods of drug delivery. Since, it is easy to manufacture the commercialization process may also become cost effective. Moreover it could also be able to cure chronic diseases like osteoarthritis, when appropriate analgesic drugs are incorporated. Now-a-days exposure to UV rays is giving way to skin cancer in people. Organogels can become a viable alternative, if anticancer agents are delivered through them which have not been tried till date.22

Etodolac is BCS Class II drug. It is a selective COX-2 inhibitor with 10-fold COX-2 selectivity over COX-1; therefore, it can be prescribed safely for the treatment of acute pain and inflammation. Etodolac possesses poor water solubility and high hydrophobicity which leads to low permeability. Etodolac cause gastric irritation and when taken orally causes constipation diarrhea, vomiting, headache dizziness, sore throat and blurred vision. Hence, limitation in formulating oral dosage forms. Such drugs pose a challenge in development of topical drug delivery system. This study was aimed to formulate and evaluate stable organogel formulations containing etodolac. Organogel are composed of sesame oil as oil phase, span 80 and tween 80 as organogelator and aqueous phase in appropriate ratio carbopol as consistency modifier methyl and propyl paraben as preservatives. Delivery of drugs using these organogel through skin increases the local/systemic delivery by different mechanisms that make them suitable vehicles for the delivery of anti-inflammatory agents. To achieve these objectives, the organogel was evaluated for the influence of pH, rheological properties, gel so transition study, spreadability, in-vitro drug release, globule size, extrudability, drug content, ex-vivo release and skin irritation study. The anti-inflammatory activity of selected etodolac containing formulation using carrageenan induced paw edema had been evaluated and compared with marketed gel formulation (Proxym gel).

Materials and Methods

Materials

Etodolac was kindly gifted by Ipca Laboratories Ltd., (Mumbai, India). Carbopol 934 was supplied from Research Lab Fine Chemicals (Mumbai, India). Span 80, tween 80, sesame oil, methyl paraben, propyl paraben, sodium hydroxide, potassium dihydrogen phosphate and methanol were supplied from Thomas Baker (Mumbai, India). All the chemicals used during study were of analytical reagent grade and used further without dilutions. Albino mice were obtained from Haffkine Institute (Mumbai, India).

Methods

Solubility

Identification of appropriate oil and surfactant that had good solubilizing capacity of etodolac and thus, could be used as the oil phase and surfactant in organogel, the solubility of etodolac in various oils and surfactants were measured.

Determination of drug solubility in different oils and surfactants

The solubility of etodolac in various oils (Sesame oil, sunflower oil, clove oil, coconut oil, soyabean oil), surfactants ( Tween 80, Span80) were determined by adding an excess amount of drug to 3 ml of selected oils, surfactants separately in 10 ml capacity stopper vials and mixed using a vortex mixer (Remi motor, Mumbai, India). The mixture vials were then shaken for 48 hrs at 40±0.50C using magnetic stirrer. Further the mixtures were kept aside for 24 hrs at room temperature to reach equilibrium. The equilibrated samples were then centrifuged at 3000 rpm for 20 min. The supernatant was taken and filtered through a 0.45 μm membrane filter. The filtrates were diluted with methanol and etodolac concentration was subsequently quantified by U.V. spectrophotometer at 225 nm.16

Construction of pseudo-ternary phase diagrams

On the basis of solubility studies, the sesame oil was selected as the oil phase. Span 80 and tween 80 were selected as surfactants and distilled water used as aqueous phase. The pseudo-ternary phase diagrams were constructed using water titration method to determine the micro emulsion region and to detect the possibility of making micro emulsions with different possible compositions of oil and surfactants. The ratios of two surfactant (Span 80: Tween 80) were chosen as 1:1, 1:2, 1:3, 2:1 and 3:1. Such mixtures were prepared. These mixtures were mixed with the oil phase to give the weight ratios of 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 and 10:90. Water was added drop by drop and stirred using a magnetic stirrer until a homogeneous dispersion or solution was obtained. After each addition, the system was examined for the physical appearance. The end point of the titration was the point where the solution becomes cloudy or turbid. Pseudo ternary plots were constructed using Chemix School Software, trial version 3.6 (Oslo, Norway), and microemulsions were prepared by ternary phase diagram.4,7,16

Formulation development of organogel

Selection of ratio of two surfactants for formulation of organogel

The five ratios like 1:1, 1:2, 1:3, 2:1 and 3:1 were prepared and plotted with pseudo ternary phase diagram by using Chemix school software v3.6 trial version. The pseudo ternary phase diagram showing larger region was selected for formulation and development of organogel.

Preparation of organogel

The eight organogel formulations were prepared and formulations that containing composition having surfactant ratio 1:3 was taken for the preparation of organogel (Figure 1). Gelling agent were screened such as Carbopol 934, Span 80 and Tween 80 for formation of gel in different concentrations of 0.5-1.5% and 8.75-41.25%. Composition of different formulation of organogel is shown in Table 1.

Figure 1: Construction of ternary phase diagram.

Accurate quantity of etodoac was weighed as per the formula. Propyl paraben was dissolved in oil. Span 80 and Tween 80 were mixed thoroughly in the proportion of 1:3 (w/w) to obtain the surfactant mixture (SM), which was used as gelator. Specified amount of the S-mix was added to the sesame oil (SO) containing propyl paraben; Etodolac was added to the mixtures of oil, surfactant mixture with appropriate percentages as described in Table 1. The above mixture was stirred on magnetic stirrer for 20 min. Subsequently, water containing methyl paraben was added drop-by-drop to the gelator solution (GS) using a burette with constant stirring on magnetic stirrer until there was a formation of organogel. Carbopol added for maintained the consistency. Thus organogel were obtained spontaneously on stirring the mixture. Depending on the composition of the GS-water mixture, the system either formed gelled structures or remained as liquid mixtures. A ternary plot depicting the proportions of SM, water and SO was prepared to figure out the compositions, which formed organogels. The final concentration of etodolac in organogels was maintained at 4 % (w/w). The Figure 2 show varying composition of prepared organogel.

Figure 2: Varying composition of prepared organogel.

Evaluation of organogel

Physical appearance

The formulated etodolac organogel formulations were observed visually for their color, homogeneity and consistency, presence of any clog and sudden change in viscosity.9

pH

The pH of all formulations was measured by digital pH meter (Systronic, Mumbai, India). The pH of organogel is also required to measure because change in pH may affect the zeta potential and finally affect the stability of products. The pH meter was calibrated before each use with standard pH 4 and 7 buffer solutions. The electrode was immersed in organogels and readings were recorded on pH meter. The measurement was performed at ambient temperature and in triplicate.4,18,23

Rheological study

Rheology is an important parameter as it affects the spreadibility and adherence of the transdermal formulations to the skin surface.

The rheogram of organogel was obtained at 25±1cC with a Brookfield Digital viscometer-LV DV E (Brookfield, Massachusetts, USA). The viscosity was measured by using spindle S-96. The viscosity of all formulations was measured at different rotational speed (rpm) i.e. at 4, 5, 6, and 10.9,6,18

Spreadability

The spreadability of the formulation was determined using an apparatus suggested by Mutimer. The apparatus consisted of two glass slides (7.5 × 2.5 cm), some of which was fixed onto the wooden board and the other was movable, tied to a thread which passed over a pulley carrying a weight. 1 gm of formulation was placed between the two glass slides. 50 gm weight was allowed to rest on the upper slide for 1 to 2 minutes to expel the entrapped air between the slides and to provide a uniform film of the formulation. The weight was removed and the top slide was subjected to a pull obtained by attaching 45 gm weight over the pulley. The time required for moving slide to travel premarked distance i.e. 7.5 cm was noted. The readings obtained were indications of relative spreadability of different formulations. Spreadability is expressed in terms of time in seconds taken by two slides to slip off from jellified organogel and placed in between the slides under the direction of certain load. Lesser the time taken for separation of two slides, better the spreadability.

It is calculated by using the formula.9,12

S = M. L / T

Where, M = Weight tied to upper slide, L = Length of glass slides, T = Time taken to separate the slides. The spreadability of different organogel formulations in comparison with marketed gel are shown in Figure 3.

Figure 3: Spreadability of different organogel formulations in comparison with marketed gel.

Extrudability test (tube test)

Extrudability test is based upon the determination of weight required to extrude 0.5 cm ribbon of organogel in 10 sec. from lacquered collapsible aluminum tube. The extrudability value was calculated using following formula

Extrudability = Weight applied to extrude organogel from tube (gm) / Area (cm2)8,21

Globule size and its distribution in organogel

Globule size and distribution was determined by Malvern Zetasizer. About 1.0 gm sample was dissolved in double distilled water and agitated to get homogeneous dispersion. Sample was injected to photocell of zetasizer. Mean globule diameter and distribution was obtained.17

Drug content determination

Drug concentration in organogel was measured by UV spectrophotometer. 1 g of the prepared gel was dissolved in 100 ml of methanol. The solution was sonicated to dissolve the drug in methanol. About 1 ml of solution was withdrawn and further diluted to 100 ml. Then absorbance was measured at 225 nm in UV/VIS spectrophotometer. The % drug content was calculated using the equation, which was obtained by linear regression analysis of calibration curve of drug in methanol.4,17

In-vitro diffusion study

In-vitro diffusion was carried out by modified Franz diffusion cell. A glass cylinder with both ends open, 10 cm height, 3.7 cm outer diameter and 3.1 cm inner diameter was used as diffusion cell. An egg membrane (soaked in phosphate buffer 24 hours before use) was fixed to one end of the cylinder with the aid of an adhesive. About 1gm of organogel was taken in the cell (donor compartment) and cell was immersed in a beaker containing 500 ml of phosphate buffer (pH 7.4) as receptor compartment. The entire surface of the cell was in contact with the receptor compartment which was agitated using magnetic stirrer and a temperature of 37±1 0C was maintained. Sample of 5 ml of the receptor compartment was removed at 1 hour interval of time over a period 8 hours with same amount replaced to maintain sink condition. The sample was analyzed at 279 nm against blank using UV Spectrophotometer. Amount of etodolac released at various time intervals was calculated with the help of calibration curve with phosphate buffer (pH 7.4) and plotted against time.4,8,23

Gel-sol transition study

Gel-sol transition temperature (Tg) was found out by incubating the organogels in a water bath, whose temperature was varied from 30-70 0C. The temperature, at which the gels started to flow, when the glass vials were inverted was noted as the gel-sol transition temperature.6,11

Ex-vivo diffusion study

The optimized formulation and the formulations giving better in vitro drug diffusion rate were selected for the ex vivo diffusion study. The wistar mice weighing average 175 ± 25 g were shaved at abdominal region. After anaesthesia to the rats, the abdominal skin was removed surgically from the animal and adhering subcutaneous fat was carefully cleaned. The dermal side of the skin was kept in contact with phosphate buffer, pH 7.4 for 2 h before start of study. Organogel formulation, 1 gm was placed on the membrane and dipped it into receptor medium and maintained the temperature at 37 ± 1 0C, aliquots of 5 ml were withdrawn at different time intervals and same volume of buffer was added to maintain sink conditions. The release profile data of prepared organogel (OG3) formulation was compared with the marketed gel (Proxym gel).3

Skin irritation study

All the materials used for the preparation of organogel fall under generally regarded as safe (GRAS) category. Concentration of all materials is very critical issue for this formulation. Large amount of surfactants is usually irritating to the skin. Therefore, skin irritation test was performed to confirm concentrations of materials used for organogel preparation is safe. In this study a set of 6 mice. The organogel (OG3) formulation was applied on the shaven skin of mice. Any skin changes i.e., change in color and changes in skin morphology were observed for a period of 24 hrs.17,10

Anti-inflammatory study

The study was conducted in accordance with the approval of the Animal Ethical Committee, MET'S Institute of Pharmacy, CPCSEA Reg. no-1344/ac/10/CPCSEA/IAEC/4. Albino mice were selected for this study. Fifteen animals were selected and weighed each individually, kept them fasted overnight and divided into 3 groups (control, standard and test), each group containing 5 animals. 0.1ml. of 1% (w/v) carrageenan was injected on the left hind paw to induce edema. About 0.5 gm of organogel formulation was applied half an hour prior to carrageenan injection and the paws thickness was measured using vernier caliper at the time interval of 0, 30, 60, 120 and 180 minute. The % inhibition of diameter paw edema by etodolac organogel and marketed etodolac gel (Proxym gel) treated group compared with carregeenan control group. From the mean edema volume, the percentage inhibition of the edema was calculated between the treated and control groups.

% Inhibition of Test ……………… (1)

% Inhibition of Standard ……………... (2)

Where, Vc = Paw Volume of Control, Vt = Paw Volume of Test, Vs = Paw Volume of Standard.

The results were analyzed statistically by one way ANOVA followed by Dunnet's test.9,10,14]

Stability studies

The prepared etodolac organogel formulations were stored away from light in collapsible tube at 40 0C and 75% RH for 3 months. After storage the samples were tested for their physical appearance, pH, % drug release, viscosity and % drug content.4,10

Results and Discussion

Selection of oils, surfactants for formulation study

Oils and surfactants were selected on the basis of results of solubility study obtained. Surfactants like Span 80, Tween 80 and oil like sesame oil had showed higher solubilizing capacity for the drug, hence selected for further study. The results are given in Table 2.

Physical appearance

The prepared organogels were evaluated for physical appearance, homogeneity, consistency and change in viscosity. The results indicated that the organogels were pale yellow in colour, homogenous without grittiness; having semisolid consistency without change in viscosity for required period. This confirms the stability of organogels.

Table 2: Solubility of etodolac in different oils and surfactants.

Oils / surfactants

Solubility (mg/ml)

Sunflower oil

15.56

Coconut oil

36.97

Soybean oil

37.57

Clove oil

54.4

Sesame oil

64.66

Span 20

67.89

Span 80

85.98

Tween 20

362.02

Tween 80

452.7

Table 3: Gel-sol transition and pH of different organogel formulations.

Formulations

pH (Mean±S.D.)

Tgs(0C)

OG 1

6.13±0.55

50

OG 2

6.01±0.44

65

OG 3

6.85±0.06

65

OG 4

5.68±0.25

45

OG 5

6.33±0.38

60

OG 6

6.07±0.45

55

OG 7

6.1±0.24

70

OG 8

5.92±0.23

70

pH determination

Since, topical systems are directly applied on the skin; their pH should be compatible with the skin pH. An acidic or basic pH causes skin irritation or disruption of the skin structure. pH of all formulations were found to be between 6-6.8 summarized in Table 3 which is acceptable for skin preparations.

 

Gel-sol transition analysis

The organogels were subjected to increasing temperature starting from 30 0C. An increment of 5 0C was made after 5 min of incubation at the previous temperature. The samples were considered to have undergone gel-sol transition, when they started to flow, the gel-to-sol transition temperature of the organogels varied from 40 0C to 70 0C, depending on the composition of the organogels shown in Table 3. As the temperature increased, there was a corresponding increase in the surface free energy with the subsequent increase in mobility of the self-assembled structures formed by the gelators. With further increase in temperature, the interactions amongst the self-assembled structures gets reduced which leads to the disruption of networked structure, thereby causing the gelled system to flow freely.

In general, the gel-sol transition was found to be >60 0C. Any deviation from this leads to the decrease in the Tgs, indicating that the proportions of S-mix and water plays an important role in the formation of a thermodynamically stable gel. Gel-Sol Analysis. (a) Sample OG3 at 30 0C and (b) Sample OG3 at 65 0C are shown in Figure 6.

Figure 4: Comparison pattern of cumulative amount of etodolac diffused from organogel formulations.

Figure 5: Zero ordered kinetic treatment for organogel containing etodolac.

Rheological study

Rheological behavior of the organogel indicated that the systems were showed non-Newtonian shear thinning pseudo plastic type of flow, i.e. shear thinning in nature showing decrease in viscosity at the increasing shear rates. The gelator molecules involved in the formation of the gelled structures via fluid-filled microstructures. As the applied shear is increased, the hydrophobic interactions are not able to keep the fluid-filled microstructures together. This results in the transition of the system from the gelled phase to the free-flowing liquid phase, marked by the disruption of the 3-dimensional networked structures. An increase in the concentration of carbopol 934 and water (0.5 to 1.5%) were expected to show increase in viscosity.

(a)(b)

Figure 6: Gel-Sol Analysis. (a) Sample OG3 at 30 0C and (b) Sample OG3 at 65 0C.

All formulations exhibited shear thinning properties. The results are given in Table 4.

Spreadability study

One of the essential criteria's for an organogel that it possesses is its good spreadability. A more viscous formulation would have poor spreadability. Spreadability is a term expressed to denote the extent of area to which gel readily, and spreads on application to the skin. The therapeutic efficacy of a formulation also depends upon its spreading value. The spreadability of different organogel formulations is shown in the Figure 3.

From the result obtained it was observed that the OG3 formulation shows the more spreading coefficient as compared to other formulations shown in Table 5. OG3 formulation gives the spreading coefficient 6.45±0.055 gm.cm/sec which may be due to presence of optimum concentration of gelling agent.

Extrudability study

Extrudability of the organogel is depending upon the viscosity of that organogel. Less viscid the organogel, lesser the force required to remove it from tube, thus shows better extrudability. The extrudability of formulated organogels is showed in Table 5.

From the obtained data it can be concluded that, the formulations OG1 and OG7 was contained highest amount of carbopol 934 i.e.1.5%, so it showed lowest extrudability due to highest viscosity among all formulations. OG2 and OG3 had showed best extrudability than other formulations as it contained carbopol 934 in 0.5%. Other formulations also showed good extrudability due to less composition of gelling agent.

Globule size and its distribution in organogel

From result we can conclude that globule size in all organogel formulation decreases when the concentration of surfactant and co-surfactant decrease. As compared with globule size of micro emulsion with organogel there is slight increase in globule size of organogel may be due to effect of addition of gelling agent into it, due to gelling agent might have led to the entrapment of oil globule in its network thus, causing slight increase the interfacial tension between oil water phases. Globule size distribution of formulations showed in Table 5.

Drug content determination

The drug content of all formulations was found to be in the range of 96.55±1.6 % to 99.78±0.87% given in Table 5. Hence uniformity of drug content was found satisfactory and was within Pharmacopoeial limits i.e. 95.0 to 105.0 % of etodolac according to British Pharmacopoeia, 2005.2

In-vitro diffusion study

In-vitro release profiles of etodolac from its various organogel formulations are represented in Table 5. It was observed that all formulations were become swelled at the end of experiment due to penetration of diffusing media into gel matrix which cause breaking of gel matrix, thus, release of drug. Comparison pattern of cumulative amount of etodolac diffused from organogel formulations are shown in Figure 4. The higher drug release was observed with formulations OG2 and OG3. This may be due to presence of minimum amount of gelling agent i.e. carbopol 934. The minimum amount of carbopol 934 cause less viscous formulation as compared to other organogel formulations, leads to less packed gel matrix which easily get broken, thus higher release of drug. The formulations OG2 and OG3 showed 93.71% and 96.36% cumulative drug release at the end of 8 hr. respectively and these are shown in Table 5. Initial burst release followed by control release was observed for all formulations. When data was plotted according to zero-order kinetics linear plots were obtained. Highest regression coefficient values were obtained for zero-order the values ranging from 0.965 to 0.989, suggesting the mechanism of release from all the formulations followed Zero-order kinetics as shown in Figure 5.

Table 7: Percent cumulative drug release from organogel formulation and marketed gel.

Time (Hr.)

 

% Cumulative Drug Release

OG 3

Marketed Gel

1

2.31

0.92

2

9.74

7.87

3

22.34

13.97

4

32.28

25.68

5

46.95

31.5

6

54.36

39.21

7

63.23

48.86

8

73.1

59.52

Ex-vivo diffusion study

Ex-vivo diffusion study was performed using fresh mice skin. The diffusion study was performed on both standard marketed etodolac gel and formulated etodolac organogel (OG3). The % drug release of both marketed gel and organogel are given in Table 7.

The standard etodolac gel showed 59.52% permeation of etodolac through mice skin at the end of 8 hrs, whereas formulated organogel i.e. OG3 showed permeation of etodoac was 73.10% at 8 hrs. The OG3 batch contains carbopol 934 in the concentration of 0.5% w/w, at this concentration carbopol 934 possess lower viscosity and thus imparts in better diffusion of drug through loosely bound network of gel, provide good release of drug and thus increasing permeation. Both of these figures indicated that formulated organogel gives higher flux and permeation as compared to standard marketed gel.

Table 8: Skin irritation study of organogel formulation using mice skin.

No. of mice

 

Hours

Erythema

Edema

1

 

2

_

_

2

 

4

_

_

 

3

 

6

_

_

 

4

 

8

_

_

 

5

 

24

   
 

_

_

The enhanced permeation from the organogel due to the smaller droplet size of the micro emulsion, etodolac directly diffuse from the droplets to the stratum corneum without micro emulsion fusion to the stratum corneum and subsequent permeation enhancement. The results were analyzed statistically by ANOVA followed by paired t-test indicating significant change in the ex-vivo release (P<0.0001). % Cumulative amount of etodolac diffused through mice skin using modified diffusion cell are shown in Figure 7.

Skin irritation study

Skin irritation test of etodolac organogel formulation was performed on mice. They were kept under observation for 24 hrs. The observations were recorded on the basis of intensity of signs for each animal is as shown in Table 8.

After follow up of observation for 24 hrs, formulation did not showed any evidences of skin irritation such as redness of skin or inflammation at the site of application. Thus, it may be concluded that selected formulation doesn't cause any irritation reaction, hence considered as safe for topical application.

In-vivo anti-inflammatory study

The anti-inflammatory study was performed by using three groups namely control, standard and test. Each group contains five mice. The standard group treated with carrageenan and etodolac (proxym) gel, control was treated with carrageenan only while test was treated with carrageenan and organogel (OG3) formulation. Paw volume was measured using vernier caliper up to 3 hrs after treatment and % inhibition in paw volume was calculated. It is given in Table 9.

Figure 7: % Cumulative amount of etodolac diffused through mice skin using modified diffusion cell.

The mean ±SEM values of paw diameter of three groups represented in Table 10. Control group remained untreated so the paw volume goes on increasing after interval of 3 hrs which showed inflammation caused by carrageenan injection. Standard and test group contains carrageenan along with etodolac standard gel (Proxym gel) and formulated organogel (OG3) respectively. Both of the groups were showed decrease in paw volume as time proceeds indicates anti-inflammatory activity of both standard and test groups. % inhibition of paw volume in standard and test groups are 68.94 and 51.70 % which indicates that formulated organogel was as effective as standard gel in its anti-inflammatory activity.

The P value is <0.0001 is considered as extremely significant by applying ANOVA test followed by Dunnett test.

Stability study

Short term accelerated stability study was performed at 40 0C and 75 % RH for 3 months. After the period of 3 months the organogel (OG3) formulation was tested for its physical appearance, pH, drug content, viscosity and drug release. After performing tests organogel formulation was found to be translucent pale yellow and homogeneous. The pH, drug content, viscosity and drug release of formulation was found to be 6.38±0.40, 52470 cps, 98.29±2.5%, and 91.1±1.54% respectively Table 10 indicating the prepared formulation was stable for the storage period.

Conclusions

The current study deals with the development of Span 80-Tween 80 based organogels. Amongst all formulations, organogel prepared with oil (5%), Smix (50%), carbopol 934 (0.5%) and water (45%) were better with respect to overall product qualities. When organogel was compared with the marketed gel, drug release from the organogel was found to be increased and prolonged. Reduction of dose of etodolac thus, formulating cost effective formulation. The formulations followed zero ordered kinetic model of drug release which involves control release. In-vitro diffusion studies indicated that the organogels may be used as matrix for controlled delivery systems. After performing anti-inflammatory study, it was found that the anti-inflammatory activity of formulated organogel (OG3) was as effective as standard marketed etodolac gel. The formulated organogel showed no irritation after performing skin irritation study using mice. The stability of the organogels was found to be dependent on the SM and water proportions. The results indicated that the developed matrices may be used as a vehicle for controlled delivery system. Thus, results of the current study clearly indicated a promising potential of the etodolac organogel as an alternative to the conventional dosage form. However, further clinical studies are needed to assess the utility of this system. By considering all above points it was concluded that, the objective of the present research study can be achieved successfully.

 

Acknowledgements

The authors are thankful to IPCA Laboratories LTD., Mumbai for generously providing gift sample of Etodolac. Also thankful to the Trustees, MET's Institute of Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik for providing excellent facilities.

Funding: No funding sources

Conflict of interest: None declared

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