Research Article

Microemulsion-Based Topical Hydrogels of Tenoxicam for Treatment of Arthritis

Shishu Goindi,1,2 Manleen Narula,1 and Atin Kalra1

Received 23 April 2015; accepted 3 August 2015; published online 19 August 2015

Abstract. Tenoxicam (TNX) is a non-steroidal anti-inflammatory drug (NSAID) used for the treatment ofrheumatoid arthritis, osteoarthritis, ankylosing spondylitis, backache and pain. However, prolonged oraluse of this drug is associated with gastrointestinal adverse events like peptic ulceration, thus necessitatingits development as topical formulation that could obviate the adverse effects and improve patientcompliance. The present study was aimed at development of microemulsion-based formulations of TNXfor topical delivery at the affected site. The pseudoternary phase diagrams were developed andmicroemulsion formulations were prepared using Captex 300/oleic acid as oil, Tween 80 as surfactantand n-butanol/ethanol as co-surfactant. Optimized microemulsions were characterized for drug content,droplet size, viscosity, pH and zeta potential. The ex vivo permeation studies through Laca mice skin wereperformed using Franz diffusion cell assembly, and the permeation profile of the microemulsion formu-lation was compared with aqueous suspension of drug and drug incorporated in conventional cream.Microemulsion formulations of TNX showed significantly higher (p<0.001) mean cumulative percentpermeation values in comparison to conventional cream and suspension of drug. In vivo anti-arthritic andanti-inflammatory activity of the developed TNX formulations was evaluated using various inflammatorymodels such as air pouch model, xylene-induced ear edema, cotton pellet granuloma and carrageenan-induced inflammation. Microemulsion formulations were found to be superior in controlling inflammationas compared to conventional topical dosage forms and showed efficacy equivalent to oral formulation.Results suggest that the developed microemulsion formulations may be used for effective topical deliveryof TNX to treat various inflammatory conditions.

KEY WORDS: arthritis; dermal delivery; microemulsion; NSAID; tenoxicam.

INTRODUCTION

Tenoxicam (TNX) is an analogue of a piroxicam and isused to relieve inflammation, swelling, stiffness and pain asso-ciated with rheumatoid arthritis, osteoarthritis, ankylosingspondylitis, backache etc. It is commercially available in sev-eral dosage forms such as tablets, suppositories and injectableswith each dosage form having its own limitation pertaining topatient compliance (1).

Like other non-steroidal anti-inflammatory drugs(NSAIDs), oral administration of TNX shows gastrointestinaladverse effects with detrimental effects not only limited to theoesophagus and stomach but to the small intestine as well (2).Moreover, the chronic oral use of NSAIDs has been found toincrease the risk of peptic ulcer disease complications by 3–5-fold, and it has been estimated that 15–35% of all peptic ulcercomplications is a consequence of prolonged NSAIDs use (3).Therefore, topical administration of NSAIDs is becoming animportant alternative to oral route of administration (4). Ad-ministered topically, NSAIDs penetrate slowly and in smallquantities into the systemic circulation; bioavailability and

maximal plasma NSAID concentrations after topical applica-tion are generally less than 5 and 15%, respectively, comparedwith equivalent oral administration (5). Common topical dos-age forms for administration of NSAIDs include creams, gelsand ointments. However, these primitive dosage forms sufferfrom inefficient percutaneous permeation through the humanskin. This has lead to development of colloidal delivery sys-tems such as liposomes, niosomes, microemulsions and solidlipid nanoparticles to improve drug permeation through theskin. In lieu of this, various novel dermal formulations of TNXhas been explored in the recent years with development oftransdermal films and proniosomal gel formulation (6,7).

Microemulsions are colloidal dispersions composed of oilphase, aqueous phase, surfactant and co-surfactant in appro-priate ratios. These consist of single optically isotropic andthermodynamically stable system with a droplet diameter usu-ally within the range of 10–100 nm (8). Because of theirstructural components, microemulsions have become increas-ingly popular due to their high solubilization potential for bothlipophilic and hydrophilic agents (9). Their ability to reducediffusional barrier of the stratum corneum coupled withsuperior thermodynamic stability help in effective cutane-ous delivery of drugs. However, they possess low viscosity;therefore, gelling agents like Carbomer 940 and xanthangum are generally added to make a suitable dosage formfor topical application (10).

1 University Institute of Pharmaceutical Sciences, Panjab University,Chandigarh, 160014, India.

2 To whom correspondence should be addressed. (e-mail:shishugoindi@yahoo.co.in)

AAPS PharmSciTech, Vol. 17, No. 3, June 2016 (# 2015)DOI: 10.1208/s12249-015-0383-0

597 1530-9932/16/0300-0597/0 # 2015 American Association of Pharmaceutical Scientists

The aim of th i s s tudy was to develop nove lmicroemulsion-based formulations for dermal delivery ofTNX and to investigate ex vivo permeation rate, anti-arthritic and anti-inflammatory efficacy of different TNXpreparations using various animal models of acute and chronicinflammation. Therapeutic efficacy of developed topical for-mulations was also compared with oral formulation.

MATERIALS AND METHODS

TNX was obtained as a gift sample from Cipla Ltd, Mum-bai, India. Captex 200 and 300 were obtained as a gift samplefrom Abitech Corporation, USA. Oleic acid, isopropylmyristate (IPM), castor oil, Tween 20 and Tween 40 wereprocured from Loba Chemie Pvt. Ltd., Mumbai, India. Tween60 and Tween 80 were purchased from S.D. Fine Chem Ltd.,Mumbai, India. All the other chemicals used were of theanalytical grade.

Animals

Male Laca mice (25–30 g) and female Sprague-Dawleyrats (200–250 g) bred at the Central Animal House (CAH) ofPanjab University, Chandigarh, India, were used for the study.Animals were fed with a standard diet ad libitum and housedin a temperature-controlled room (25±2°C, 60–70% humidi-ty), in accordance with the institution ethical committee regu-lations. Animals were acclimatized to laboratory conditionsbefore the test.

Solubility Studies

Solubility studies of TNX were carried out in differentoils (isopropyl myristate (IPM), castor oil, Captex 200, Captex300 and oleic acid) and surfactants (Tween 20, Tween 40,Tween 60 and Tween 80), using the shake flask method ofsolubility determination (11). Excess of drug was added to afixed volume of each solvent (10 mL) in different flasks, whichwere kept at 37±0.5°C in a thermostatic water shaker bath for24 h. The flasks were removed after 24 h, contents werefiltered through a 0.45-μm filter, and filtrates were analyzedspectrophotometrically at 370 nm after appropriate dilution.Studies were carried out in triplicate (12).

Selection of Microemulsion Components

Selection of oil was done based on solubility of TNX inoil. Further, surfactant was selected based on its emulsificationability. A 1:1 mixture of surfactant (Tween 80) and oil (Captex300/oleic acid) was homogenized, and 50 mg of isotropic mix-ture was weighed and diluted with double-distilled water to50 ml to give a fine emulsion. The emulsions were allowed tostand for 2 h and their transmittance was determined at650 nm by UV spectrophotometer using double-distilled wateras blank.

Titration method was used to find the appropriate co-surfactant (13). The mixtures of surfactant and co-surfactantwere made in predetermined weight ratios (Km). Each surfac-tant and co-surfactant mixture was added to mixtures of oiland water in a dropwise manner under the mixing of magneticstirrer, at room temperature. After each addition, the samples

were stirred to reach the equilibrium (water/oil ratio was keptconstant at 1:1). When a sufficient amount of the surfactant/co-surfactant mixture (S/Cos) was added to completely solu-bilize the equal masses of oil and water (SCoSmin), a singlephase microemulsion was formed. The transparent sampleswere allowed to equilibrate for minimum of 72 h.

Construction of Ternary Phase Diagrams

In order to ascer ta in microemuls ion reg ion ,pseudoternary phase diagrams were constructed using themethod of titration (14). The mixture of oil (Captex 300/oleic acid) and series of S/Cos mixtures were titrated withdouble-distilled water at room temperature under moderatemagnetic stirring. After being equilibrated, the mixtures wereassessed visually and determined as being microemulsions,emulsions or gels.

Preparation of Microemulsion Formulations

After identification of the microemulsion region in thephase diagram, TNX loaded o/w microemulsion (TNX 03 and04) were prepared by dissolving TNX (0.15%) in Captex 300/oleic acid. Subsequently, a mixture of surfactant (Tween 80)and co-surfactant (n-butanol/ethanol) was added to the oilyphase under continuous magnetic stirring and appropriateamount of water was added dropwise, and a clearmicroemulsion was obtained spontaneously (Table I). Finally,both the microemulsion formulations were gelled withCarbopol 940 in order to make them rheologically favourablefor topical delivery.

Preparation of Conventional Cream and Suspension

TNX (0.15%) was formulated in a conventional cream(TNX 01) containing sorbitan mono-oleate (6%), white beeswax (3%), white soft paraffin (36%) and liquid paraffin(15%). TNX suspension (0.15%) (TNX 02) was prepared bydispersing TNX in 4% w/v sodium carboxymethylcellulose(CMC) dispersion in water.

Characterization of Microemulsions

Prepared microemulsion formulations were characterizedfor drug content, pH, droplet size, zeta potential and viscosity.Morphology and structure of the microstructures of drug-loaded microemulsion was determined with the aid of trans-mission electron microscopy (TEM) (15). Droplet size andsize distribution were analyzed using laser light scatteringtechnique (Malvern Zetasizer™).

The rheo log i ca l behav iou r o f the p reparedmicroemulsion was determined with a DV-II viscometer(Brookfield, UK). The microemulsions were submitted to upand down cycles (0–100 rpm spindle rotation speed) at 32.0±0.1°C, and the rheological behaviour was evaluated by plot-ting shear rate against shear stress (16). The spreadability ofthe developed TNX formulations was evaluated using TextureAnalyzerTM equipment, equipped with a 5-kg load cell todetermine different rheological properties of prepared formu-lations viz., work of shear and stickiness.

598 Goindi et al.

Ex Vivo Skin Permeation Study

Male Laca mice skin (thickness 0.5–0.7 mm) previouslyshaved and washed with normal saline was mounted on Franzdiffusion cell assembly with the stratum corneum facing donorcompartment. The receptor compartment consisted of 30 mLphosphate buffer (pH 6.4 with 20% ethanol) maintained at 37±1°C and diffusional area of 2.84 cm2. TNX showed sufficientsolubility (1.044±0.041 mg/ml) in this media. For ex vivo per-meation studies, 1 g of formulation TNX 01/TNX 02/TNX 03/TNX 04 containing 1.5 mg of TNX was applied on the skin inthe donor compartment. An aliquot of 1-mL sample waswithdrawn at suitable time intervals and replaced immediatelywith an equal volume of fresh diffusion medium (17). Thesamples were analyzed spectrophotometrically at 370 nm aftersuitable dilutions. Similarly, permeation studies were per-formed using blank formulations without drug. Blank valueswere subtracted from test to account for any absorbancevalues due to leachants from the skin or formulation compo-nents. All experiments were performed in triplicate.

At the end of the permeation study, the skin mounted onthe diffusion cell was removed carefully. The remaining for-mulation in the donor compartment adhering to the skin wasscraped off. The above-mentioned cleaned skin was washedthree to four times with distilled water. Subsequently, the skinwas dipped in 5 ml methanol and kept overnight in a refriger-ator (18). Supernatant was filtered using a 0.45-μm filter, andthe filtrate was analyzed spectrophotometrically at 370 nm fordrug concentration after suitable dilution (19). Each experi-ment was conducted in triplicate.

Histopathology of Skin

Laca mice were used in the study. The hair on the dorsalside of the mice was removed in a tail-to-head direction with-out damaging the skin. Microemulsion formulations withoutTNX were applied uniformly on the dorsal region and allowedto be kept in contact for 4 h. Subsequently, the animals weresacrificed by cervical dislocation, the dorsal area was cut andsubcutaneous tissue was removed. Each specimen was thenfixed in 10% buffered formalin, embedded in paraffin andmicrotome. The specimens were stained with haematoxylinand eosin and observed under a high-power light microscope(20).

Thermodynamic Stability Studies

Thermodynamic stability of prepared microemulsion for-mulations was evaluated by a previously reported method(21).

Heating-Cooling Cycle

Six cycles between refrigerator temperature 4 and 45°Cwith storage at each temperature for 48 h were studied.

Centrifugation

The formulations passed in previous step were centri-fuged at 3500 rpm for 30 min.

Freeze-Thaw Cycle

Three freeze-thaw cycles between −20°C and +25°C withstorage at each temperature for 48 h were done for theformulations.

Temperature Stress Studies

Microemulsion formulations were stored in sealed glasscontainers at 4, 25 and 40°C for 90 days. The stability of themicroemulsions was checked periodically with respect totransparency, phase separation, colour change and drug con-tent of TNX at three different temperature conditions.

PHARMACODYNAMIC EFFICACY

Air Pouch Model for Evaluating Anti-Arthritic Activity

Anti-arthritic activity of TNX formulations was evaluatedby air pouch formation in female rats (Sprague-Dawley)weighing 200–250 g each. Animals were divided into sixgroups of three rats each with each group receiving differenttreatment (control, TNX 01, TNX 02, TNX 03, TNX 04 andoral). Dorsal air pouch were prepared by subcutaneous injec-tion of 10 ml of air through a sterile syringe. Three days afterthe first injection of air, the pouches were reinflated by

Table I. Composition of TNX Microemulsions

Formulation Captex 300 Oleic acid Tween 80+ethanol (1:1) Tween 80+butanol (2:1) Water

TNX 03 10% 45% 45%TNX 04 10% 55% 35%

Table II. Solubility of TNX in Different Oils and Surfactants

Solvent system Solubility (mg/ml)

OilsIsopropyl myristate 1.276±0.158Oleic acid 3.194±0.042Captex 300 2.933±0.158Captex 200 2.537±0.042Castor oil 0.112±0.011

SurfactantsTween 80 12.410±0.053Tween 60 9.276±0.074Tween 40 4.813±0.074Tween 20 7.238±0.148

599Microemulsion Formulations of Tenoxicam

injection of an additional 10 ml of air (22,23). On the sixth day,1 mL of 1% carrageenan in saline was injected into the pouch.Different TNX formulations (0.15%) were topically appliedtwice daily. After 48 h, the animals were sacrificed with over-dose of ether, their pouch were flushed with 1 mL of saline.The pouch fluid was collected and immediately cooled in iceand evaluated for pouch volume, granuloma tissue weight andnumber of white blood cells.

Xylene-Induced Ear Edema

The effect of the different TNX formulations on acutetopical inflammation was evaluated by a xylene-induced earedema in mice (24). Adult male Laca mice weighing 25–30 gwere divided into six groups of four animals each with eachgroup receiving different treatment (control, TNX 01, TNX02, TNX 03, TNX 04 and oral). Different TNX formulations(0.15%) were applied on the outer surface of the right ear.The control group received plain carbopol gel. After 30 min ofadministration of test formulations, topical inflammation wasinduced on the dorsal surface of the right ear by application ofxylene (20 μl). One hour after induction of inflammation, themice were killed by overdose of ether anaesthesia and bothears were removed. Circular sections (7 mm diameter) of boththe right (treated) and left (untreated) ears were punched outusing a borer, and weighed.

Edema was determined as the weight difference betweenthe two plugs. The anti-inflammatory activity was evaluated as

percent edema inhibition in the treated animals relative tocontrol animals.

% Edema inhibition ¼ control − testcontrol

� �� 100 ;

where control=difference in mean weight of the right ear plugof control animals and mean weight of the left ear plug ofcontrol animals; test=difference in mean weight of the rightear plug of treated animals and mean weight of the left earplug of treated animals (25,26).

Cotton Pellet Granuloma Test in Rats

Chronic inflammation was evaluated using cotton pelletgranuloma test in rats (27). Adult female Sprague-Dawley ratsweighing 250–300 g were divided in six groups of three ratseach with each group receiving different treatment (control,TNX 01, TNX 02, TNX 03, TNX 04 and oral). The animalswere anaesthetized with ether after shaving the fur, and sub-cutaneous tunnels were formed by a blunted forceps and20 mg of sterile cotton pellets were inserted subcutaneouslyon the dorsal side, one in each axilla. On day 1, both conven-tional and novel TNX formulations (0.15%) were topicallyapplied. The control group was kept devoid of any kind oftreatment. Formulations were administered once daily for thenext 7 days. On day 8, animals were sacrificed by overdose of

Fig. 1. The pseudoternary phase diagram of the microemulsion composed of a Captex 300,Tween 80 and n-butanol (2:1) and water b oleic acid, Tween 80 and ethanol (1:1) and water

Fig. 2. TEM of microemulsion formulations. a TNX 03. b TNX 04

600 Goindi et al.

ether. The pellets were dissected out, freed of tissue attach-ments and the wet weight of cotton pellets was determined.These were then dried in the oven overnight at 70°C andreweighed. The percent inhibition was expressed by compar-ing the mean weight in the test groups with that in the control.

Suppression of Carrageenan-Induced Inflammation

The anti-inflammatory activity was conducted on Sprague-Dawley rats against the carrageenan-induced rat paw edema(28). The animals were divided into six groups consisting ofthree animals each with each group receiving different treat-ment (control, TNX 01, TNX 02, TNX 03, TNX 04 and oral).Different TNX formulations (0.15%) were applied topically halfan hour prior to the administration of carrageenan. The controlgroup received the same experimental handling as test groupexcept that vehicle alone was applied topically. After 30 min,0.05 ml of 1% carrageenan was injected into the right hind footof each rat under the planter aponeurosis (29). The volume ofthe paw of rats of control (Vc) and treated (Vt) groups atdifferent intervals of time was measured by a plethysmograph.The percent inhibition of edema volume was calculated usingthe following formula (30).

%Inhibition ¼ Vc−Vt

Vc� 100

Statistical Evaluation of Data

All data was statistically analyzed by one-way analysis ofvariance (ANOVA). Results were quoted as significant wherep<0.001.

RESULTS AND DISCUSSION

Screening of Components—Solubility Studies

From different oils screened, solubility of TNX was foundto be maximum in oleic acid followed by Captex 300, Captex200, isopropyl myristate and castor oil, respectively. Amongstthe surfactants screened TNX showed maximum solubility in

Tween 80 followed by Tween 60, 20 and 40, respectively(Table II).

Phase Studies

In most cases, single-chain surfactants alone are unable toreduce the o/w interfacial tension sufficiently to form amicroemulsion (31,32). Surfactants like Tweens require thepresence of other surface active agents, also known as co-surfactants, to form microemulsions (33). Addition of shorterchain co-surfactant gives positive curvature, thereby favouringformation o/w type microemulsion. Amongst all the surfac-tants screened, Tween 80 showed maximum % transmittancewhich demonstrated its ability to emulsify the selected oils.The minimum concentration of Tween 80/co-surfactant mix-ture required to produce clear microemulsions (S/CoSmin) wasdetermined by varying surfactant/co-surfactant mass ratio(Km). For Captex 300 as oil phase, Tween 80 and n-butanol(2:1) required the minimum values in terms of percentage ofsurf/co-surf mixture used to form a clear microemulsion. Sim-ilarly for oleic acid as oil phase Tween 80 and ethanol (1:1)formed a clear microemulsion. Based on the results obtained,microemulsion region was determined with the help ofpseudoternary phase diagrams and selected formulationsTNX 03 and TNX 04 consisted of oil/surfactant/water weightratio of 10:45:45 and 10:55:35, respectively (Fig. 1).

Fig. 3. Size distribution of microemulsion formulations. a TNX 03. b TNX 04

Fig. 4. Ex vivo permeation profile of TNX cream (TNX 01), aqueoussuspension (TNX 02) and microemulsion gels (TNX 03 and TNX 04)

(mean±SD; n=3)

601Microemulsion Formulations of Tenoxicam

Characterization of Prepared Microemulsion

The results of our study indicate development of success-ful microemulsion formulations of TNX with optimum char-acteristics. The drug content of selected formulations TNX 03and TNX 04 was found to be 99.403±0.086 and 99.801±0.149,respectively. The pH of microemulsion formulations TNX 03and TNX 04 was found to be 5.54 and 5.68, respectively.Viscosity of TNX 03 was 11,928.88 cp, whereas viscosity ofTNX 04 was found to be 11,122.43 cp. TEM confirmed thespherical nature of droplets (Fig. 2), and the average diameterof droplets was found to be 106 nm for TNX 03 and 122 nm forTNX 04. The small diameter of both formulations (Fig. 3)could be attributed to the effect of co-surfactant moleculeswhich lower the fluidity and surface tension of the interfacialfilm, thereby decreasing the radius of nanodroplets (32). Zetapotential of both microemulsion formulations was found to benear zero. This could be attributed to non-ionic nature ofTween 80 which does not impart any charge to the droplet,thus providing better stability to the microemulsion system.

Both microemulsion formulations after gelling demon-strated pseudoplastic and shear-thinning behaviour. Thispseudoplasticity results from a colloidal network structure thataligns itself in the direction of shear, thereby decreasing theviscosity as the shear rate increases (16). The shear-thinningbehaviour is usually preferred so that the applied skin systems

would be stable at rest; nevertheless, upon application on theskin, the viscosity decreases and drug permeation is enhanced(34). Rheological properties play important role in efficientperformance of a topical formulation. Optimum rheologicalproperties are imperative for uniform and easy application oftopical formulation for better patient compliance. Differentparameters studied included work of shear and stickiness.Work of shear for TNX 03 and TNX 04 was found to be0.006 and 0.007 kg sec, respectively, and stickiness of−0.014 kg for TNX 03 and −0.016 kg for TNX 04, therebysuggesting non-stickiness and acceptable spreadability offormulations.

Ex Vivo Permeation Studies

The ex vivo permeation profiles of all TNX formulationsthrough mice skin are shown in Fig. 4. The cumulative amountof TNX permeated in 24 h from aqueous suspension (TNX 02)was only 7.31±0.63%, whereas permeation from conventionalcream (TNX 01) was found to be 27.972±0.81%; and frommicroemulsions, still higher skin permeation of 64.647±0.97%(TNX 03) and 70.829±0.84% (TNX 04) was obtained. Similarly,flux values from microemulsion formulations TNX 03 and TNX04 were found to be 16.100±0.26 and 12.167±0.11 μg/h/cm2,respectively, in comparison to flux values of 6.933±0.231 and1.100±0.100 μg/h/cm2 depicted by TNX 01 and TNX 02,

Fig. 5. Histopathology of a control, b TNX 03 (without drug) and c TNX 04 (without drug)

Table III. Effect of Different Formulations on Mean Fluid Volume, Granuloma Weight and Leucocytes Count in Rat Air Pouch Model

Group Mean volume of pouch fluid (ml) Mean granuloma tissue weight (g) Mean leucocytes

Control 5.43±0.404 1.506±0.074 10,866.66TNX 01 4.83±0.305 1.261±0.066 9733.33TNX 02 4.97±0.416 1.392±0.040 10,200.00TNX 03 3.51±0.458 0.689±0.032 8066.66TNX 04 3.17±0.352 0.634±0.051 7500.00Oral 2.33±0.473 0.639±0.057 6083.33

602 Goindi et al.

respectively. The results obtained are consistent with previousstudies concurring the efficacy of microemulsion in dermal deliv-ery of drugs (10). The ability of microemulsions to reduce theinterfacial tension between the skin and vehicle results in fasterpermeation. The presence of known permeation enhancer n-butanol in TNX 03 also explains its better permeation. Theintercalation/partition of the short-chain n-alkanols like n-butanol into the intercellular lipids of the stratum corneum isbelieved to be a critical part of the mechanism of action of pene-tration enhancement, thus altering the diffusivity of the stratumcorneum towards TNX (35,36). Amongst the two microemulsionformulations, better permeation shown by TNX 04 might be dueto synergistic action of ethanol and oleic acid.

The skin retention of TNX from bothmicroemulsion formu-lations was superior and significantly higher (p<0.001) in com-parison to conventional cream and aqueous suspension. TNX

retention from TNX 03 was found to be 11.429±0.399%, whichwas 4.6 timeswhen compared to TNX01 (2.469±0.24%) and 11.5times when compared to TNX02 (0.988±0.18%).Whereas, TNX04 showed skin retention of 13.551±0.25%, which was 5.5 timeswhen compared to conventional cream TNX 01 and 13.7 timeswhen compared to TNX 02. The high skin retention frommicroemulsions could be attributed to microdepot formed bythem leading to higher skin retention of drug.

Histopathology

The histology of excised mice skin of control and micetreated with microemulsions (without TNX) after 4 h is shownin Fig. 5. Both microemulsions did not show any effect on themicroscopic structure of the skin and no pathological changes

Fig. 6. Representative photographs showing the inside of the pouch from different groups showing intensity of erythema andinflammation. a Control. b TNX 01. c TNX 02. d TNX 03. e TNX 04. f Oral treatment

Fig. 7. Effect of different TNX formulations on granulomatous tissue formation

603Microemulsion Formulations of Tenoxicam

were found, thus establishing the safety of the preparedformulations.

Stability

Physical appearance of microemulsion formulations aftersix heating-cooling cycles remained unchanged and the for-mulations maintained transparency. No phase separation wasobserved and formulation was found to be stable. Centrifuga-tion at 3500 rpm for 30 min did not reveal any signs of phaseseparation or precipitation, indicating physical stability ofmicroemulsion. Finally, both TNX 03 and TNX 04 were foundto be resistant to freeze-thaw cycles in terms of transparencyand phase separation.

Temperature Stress Studies

Stability studies data at the end of 90 days showed thatselected microemulsion formulations were stable for drugcontent, pH and characteristics like viscosity, clarity and col-our, thereby suggesting the long-term stability of preparedformulations.

ANTI-INFLAMMATORY EFFICACY

Anti-Arthritic Activity Using Air Pouch Model

Formation of an air pouch induces the proliferation ofcells that stratify on the inner surface of the pouch to form astructure similar to the synovial (37). Injection of carrageenaninduces inflammation and the pouch serves as a reservoir ofcells and mediators that can be easily measured in the fluidthat accumulates locally and extent of inflammation beingdirectly proportional to the volume of exudates (38). As evi-dent from results given in Table III, TNX 03 and TNX 04showed significantly higher (p 0.001) anti-inflammatory effi-cacy as compared to TNX 01 and TNX 02. Mean granulomatissue weight in the groups treated with conventional cream(TNX 01) and aqueous suspension (TNX 02) was found to betwice in comparison to group treated with microemulsion gelsTNX 03 and TNX 04. Mean leukocyte count, another mani-festation of inflammation was also significantly higher ingroups treated with conventional dosage forms (Table III).Visual examination of the inside of the air pouch revealed ahighly intense erythema and inflammation in control group,whereas in groups treated with conventional dosage forms the

Fig. 8. Effect of different TNX formulations on carrageenan-induced paw edema in rats

Fig. 9. Effect of different TNX formulations on xylene-induced ear edema in mice

604 Goindi et al.

intensity of erythema was less when compared to controlgroup (Fig. 6a–c). In comparison, the groups treated withmicroemulsions TNX 03 and TNX 04 and oral treatmentgroup showed only very slight erythema (Fig. 6d–f). Resultssuggest that microemulsions TNX 03 and TNX 04 were moreeffective than conventional topical formulations and showedefficacy comparable to oral dosage form.

Cotton Pellet Granuloma in Rats

The cotton pellet granuloma is a model of chronic inflam-mation, and dry weight has been shown to correlate with theamount of granulomatous tissue formed (39). The method isrobust and reproducible and the implanted pellet is capable ofproducing a large effect on the proliferate component of theinflammatory process (40). The granuloma is characterized bythe formation of a fibrous vascularized capsule containingfibroblast and mononuclear infiltrate cells. Decreased granu-loma tissue formation, prevention of occurrence of the colla-gen fibres and suppression of mucopolysaccharides areindicators of the anti-proliferative effect of NSAIDs (41).Daily topical application of the microemulsion formulationsTNX 03 and TNX 04 significantly (p<0.001) inhibited granu-lomatous tissue formation, in comparison to TNX 01, TNX 02and control (Fig. 7). The data also demonstrated that the anti-inflammatory activity of topical microemulsions (TNX 03 andTNX 04) was comparable to oral treatment. The high anti-inflammatory efficacy depicted by microemulsion formula-tions could be attributed to their ability to permeate anddeposit in the skin, thereby providing a means for bettercontrol of inflammatory response in comparison to conven-tional cream and suspension dosage forms.

Carrageenan-Induced Hind Paw Edema in Rats

Carrageenan is a sulphated cell wall polysaccharide foundin certain red algae. Upon injection into rat paws, carrageenanprovokes a local, acute inflammatory reaction, which is usedfor evaluating anti-inflammatory agents (42). Treatment withmicroemulsion formulations TNX 03 and TNX 04 displayedsignificant (p<0.001) inhibition of edema, 64.54 and 67.89%,respectively, in comparison to 21.57 and 6.31% showed byTNX 01 and TNX 02 (Fig. 8). The higher anti-inflammatoryefficacy of TNX 03 and TNX 04 could be attributed to itsability to get retained in skin for longer duration, thus modu-lating superior anti-inflammatory response in comparison toconventional cream and suspension.

Ear Edema Induced by Xylene in Mice

Xylene causes instant irritation of the mouse ear, whichleads to fluid accumulation and edema, characteristic of theacute inflammatory response (26). The xylene-induced mouseear edema method has certain advantages of having goodpredictive value for screening anti-inflammatory agents. Ap-plication of microemulsion formulations TNX 03 and TNX 04significantly (p 0.001) inhibited xylene-induced ear edema ascompared to TNX 01 and 02 (Fig. 9). Interestingly,microemulsion formulations displayed better anti-inflammatory efficacy than the oral treatment of drug. Possi-ble explanation for this could be due to the quick and instant

penetration of the microemulsion at the site of application,thereby proving better control over the acute inflammatoryresponse.

CONCLUSION

In the present study, o/w microemulsion formulations forthe dermal delivery of TNX were developed and character-ized. Ex vivo permeation rate of both microemulsion formu-lations was significantly higher when compared toconventional topical formulations TNX 01 and TNX 02(p<0.001). In addition to better permeation, microemulsionformulations elicited significant (p<0.001) anti-arthritic andanti-inflammatory efficacy in various animal models of arthri-tis and inflammation, and therapeutic response was compara-ble to oral formulation of drug. Moreover, histopathologystudies revealed dermal safety of both microemulsion formu-lations. In conclusion, our study demonstrated thatmicroemulsion formulations of TNX could be a potentialalternative to oral formulation of drug.

Conflict of Interest The authors report no conflict of interest.

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