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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Drais. World Journal of Pharmacy and Pharmaceutical Sciences SJIF Impact Factor 6.041 Volume 5, Issue 6, 308-318 Research Article ISSN 2278 – 4357 DEVELOPMENT, CHARACTERIZATION AND EVALUATION OF THE PIROXICAM NANOEMULSION GEL AS TOPICAL DOSAGE FORM Hayder Kadhim Drais* Ministry of Health and Environment, Babylon Health Directorate, Babylon, Iraq. Article Received on 13 April 2016, ABSTRACT Piroxicam is a non-steroidal anti-inflammatory drug. It is practically Revised on 01 May 2016, Accepted on 22 May 2016 insoluble in water. It is associated with many gastrointestinal unwanted DOI: 10.20959/wjpps20166-7031 effect. The objective of this investigation to prepare an evaluate nanoemulsion gel of piroxicam as a topical dosage form to increase *Corresponding Author solvability of piroxicam and decrease the gastrointestinal side effect Dr. Hayder Kadhim that lead to enhance piroxicam bioavailability and therapeutic activity. Drais The pseudoternary phase diagrams were constructed from the double Ministry of Health and distilled water, surfactant mixture (Smix) was tween 80 and ethanol at Environment, Babylon ratios of (1:1, 2:1, 3:1, and 4:1) and oil mixture. The pseudoternary Health Directorate, Babylon, Iraq. phase plot that has larger region of nanoemulsion was Smix ratio (3:1) from which take six of nanoemulsion formulas (NE1-NE6) for characterization of nanoemulsions and to formulate nanoemulsion gel formulas (NF1-NF6). The six of nanoemulsion gel formulas were evaluated. NF6 was the optimized formula and tested for stability and morphology. Atomic force microscopy (AFM) shows that the selected formula (NF6) is highly stable. It can be concluded that the selected (NF1) was an effective alternative for the topical delivery of piroxicam. KEYWORDS: Nanoemulsion, Piroxicam, Nanoemulsion gel. INTRODUCTION Piroxicam a non-steroidal anti-inflammatory drug utilized to relieve the symptoms of inflammatory arthritis such as (rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus), degenerative arthritis such as osteoarthritis, in acute gout and in post-operative pain. It is practically insoluble in water, however, its use has been www.wjpps.com Vol 5, Issue 6, 2016. 308 Drais. World Journal of Pharmacy and Pharmaceutical Sciences associated with a number of undesirable side-effects on the stomach and kidneys in addition to gastric mucosal damage.[1] Nanoemulsion (NE) which is nano scale delivery systems in has great interest as a part of nanotechnology. It is transparent with the range of particle sizes between 100 to 500 nm. The aqueous phase, oil phase, surface active agent, co-surfactant represent the composition of nanoemulsion.[2] However, the rheological properties of nanoemulsion make it unsuitable for application to the skin. When combine gelling agent with nanoemulsion, this make more desirable for transdermal delivery.[3] When gelling agent and nanoemulsion are utilized in combination form the dosage form is named nanoemulgel. This provide double release control system i.e. Emulsion and gel.[4] The solubility problem of most of the hydrophobic drugs make these drugs cannot be incorporated directly into the gel base.The nanoemulsion gel help that the hydrophobic drugs solubilized into the oily phase and then oil droplets are dispersed in aqueous phase leading to oil in water (o/w) nanoemulsion then this nanoemulsion can be incorporate into gelling agent to get nanoemulsion gel.[5] Thus, the aim of this research to formulate and evaluate nanoemulsion gel of piroxicam employed topically to increase solubility of piroxicam and decrease oral unwanted effect of piroxicam that lead to improve piroxicam therapeutic activity and patient compliance. MATERIALS AND METHODS MATERIALS Piroxicam supplied by Wadi Al-Rafidian factory for pharmaceutical products, Baghdad, Iraq. peppermint oil and ginger from BAR-SUR-loop Grasse A. M Franc. Tween 80 from SD fine Chemlimited (SDFCL) Mumbai, India. Methanol and ethanol from grin land chemical comp, United. Carbomer 934 from Hengshui Taocheng Chemicals Auxiliary Co., Ltd. China. KH2Po4 and Na2Po4 from Merck & Co., Inc. Germany. METHODS 1. FORMULATION OF PIROXICAM NANOEMULSION AND DRAWING OF PSEUDOTERNARY PHASE DIAGRAM The oil mixture was produced by mixing of ginger oil and peppermint oil in a ratio (1:2) to obtain oil mixture. The aqueous phase, oil mixture and surfactant mixture (Smix) are ingredients of the pseudo ternary phase diagram that is get by use the aqueous phase titration method. The surface active agent and co-surfactant is combined in various weight ratios (1:1, www.wjpps.com Vol 5, Issue 6, 2016. 309 Drais. World Journal of Pharmacy and Pharmaceutical Sciences 2:1, 3:1, 4:1) to obtain Smix. The piroxicam loaded in oil mixture mix with Smix in different weight ratios for each phase diagram, so that all parts of the pseudoternary phase diagram will be covered. The slow titration of double distilled water to admixture of the oil mixture and Smix was occurred to get the borderline of phases and ocular investigation were made for transparency. When there is a clear liquid to the eyes, stop further adding double distilled water and different o/w nanoemulsions are formalised. The pseudoternary phase plot was designed. Take seven formulas from pseudoternary phase diagram that has wider nanoemulsion area to prepare nanoemulsion gel of piroxicam.[6] 2. FORMULATION OF PIROXICAM NANOEMULSION GEL The aqueous phase was made by combining double distilled water and tween80. The oil mixture that is previously formulated constitute the oily phase. The oil mixture was sonicated for 15 minutes. The aqueous phase heated to 80 and oily phase to 70°C on hot plate; the oily phase gradually was poured to the aqueous phase with continuous stirring with magnetic stirrer at 2000 RPM and stirring was continued for 5-10 min until becoming cool at room temperature. The gel bases were formulate by dispersing carbopol 934 with specified concentration in double distilled water with constant stirring at a moderate speed using mechanical stirrer. Few drops of triethanolamine were added to obtain a pH of about ( 5.5). The nanoemulsion was then combine with the gel in (1:1) ratio to obtain homogenous nanoemulsion gel.[7] Table (1): The piroxicam nanoemulsion gel formulations. Formulation components (%w/w) Piroxicam Carbopol 934 Oil mix(1:2) Smix(3:1) Propylene glycol Ethanol Methylparaben Propylparaben Sodium sulphite Double distilled water up to www.wjpps.com Formulation code F1 F2 F3 F4 F5 F6 Piroxicam gel 1 1 5 35 0 0 0.02 0.02 0.1 1 1 5 32.5 0 0 0.02 0.02 0.1 1 1 5 30 0 0 0.02 0.02 0.1 1 1 5 27.5 0 0 0.02 0.02 0.1 1 1 5 25 0 0 0.02 0.02 0.1 1 1 5 22.5 0 0 0.02 0.02 0.1 1 1 5 0 5 5 0.02 0.02 0.1 100 100 100 100 100 100 100 Vol 5, Issue 6, 2016. 310 Drais. World Journal of Pharmacy and Pharmaceutical Sciences CHARACTERIZATION OF THE FORMULATED PIROXICAM NANOEMULSION MEASUREMENT OF THE GLOBULE SIZE The globule size was determined throug ABT-9000 nanolaser particle size analyzer. The average of globule size and globule size distribution plot was obtained. All samples were taken in three trails.[6] POLY DISPERSITY INDEX (PDI) MEASUREMENT It can obtained by ABT-9000 nanolaser particle size tester. It determine the uniformity of the globule size in nanoemulsion preparation.. The measurements were occuring in triplicate. PDI range from 0.0 to 1.0. As the polydispersity index value is closer to zero, the globules with high uniformity.[8] EVALUATION PIROXICAM NANOEMULSION GEL PHYSICAL APPEARANCE The prepared nanoemulsion gel formulas were visually analyzed for color, appearance and homogenicity. The tested samples were occurred in triplicate.[9] THE pH DETERMINATION The pH of all the nanoemulsion gel preparations was tested by a pH meter at room temperature. The human skin is naturally acidic with a normal pH of 4-6. The women have a pH more acidic skin than men.One of the high important factors in the evaluation of nanoemulsion gels is the pH. The pH values affect on ionization of the drug i.e. ionized and unionized forms of the drug. The tested samples were occurred in triplicate.[10] THE DRUG CONTENT MEASUREMENT By UV-visible spectrophotometer, the drug content was measured(6). The samples were diluted with methanol then read in the UV analyzer. The tested samples were occurred in triplicate. The absorbance was observed at 200 nm and the drug content was determined by the following equation. Drug content = (Analyzed content/Theoretical content) x 100 ….. (Eq2) SWELLING INDEX ESTIMATION Swelling index is the separation of molecules of formulas by water molecules cause an expansion in volume of the mass. It associated to application area of nanoemulsion gel. One gram of piroxicam nanoemulsion gel formulations was draped with aluminium foil that is www.wjpps.com Vol 5, Issue 6, 2016. 311 Drais. World Journal of Pharmacy and Pharmaceutical Sciences punched to get holes and put in phosphate buffer pH 6.8 for 6 hours, then the swollen samples wiped with the filter paper to eliminate access of absorbed distilled water on the surface and then it was rapidly weighed on an electronic balance. All samples were taken in three trails. The swelling index was measured by utilizing the following equation(Eq). Sw = [(Wt – Wo) / Wo] × 100….. (Eq1) Where, SW = percentage of swelling index of piroxicam nanoemulsion gel Wt = the weight (g) of the nanoemulsion gel at time t Wo = initial weight (g) of the piroxicam nanoemulsion gel.[11] IN VITRO RELEASE MEASUREMENT A United States Pharmacopeia (USP) dissolution tester apparatus II is the device that is used to measure the in vitro release of piroxicam from formulas. One gram of piroxicam nanoemulsion gel formulations (NF1-NF6) and piroxicam gel was put in class tube with 1.5cm diameter, covered with a cellulose acetate membrane which was formerly soaked in phosphate buffer of pH (5.5) for about 24 hr. The membrane adquately sealed and inverted under the surface of 900 mL of phosphate buffer of pH (5.5) containing 1% polysorbate 80, at 37 ± 0.5 °C with stirring speed of 100 RPM. The sample of 5 mL was taken at 0, 15, 30, 60, 90,120, 150, 180, 210, 240 min and replaced with an equivalent amount of dissolution medium. The drug content of the samples was determined by a UV spectrophotometer at 200 nm. The tested samples were occurred in triplicate.[12,13] ATOMIC FORCE MICROSCOY (AFM) ANALYSIS By AFM angstrom advanced inc. AA3000 USA, the morphology of selected piroxicam nanoemulsion gel formula was determined. AFM analysis was occurred by placing drops of the sample onto a glass slide and then measure.[14] STATISTICAL ANALYSIS The tested samples were occurred in triplicate and use analysis of variance (ANOVA) at level (P<0.05) to explain the results.[6] www.wjpps.com Vol 5, Issue 6, 2016. 312 Drais. World Journal of Pharmacy and Pharmaceutical Sciences RESULTS AND DISCUSSION FORMULATION OF PIROXICAM NANOEMULSION, DRAWING OF PSEUDOTERNARY PHASE DIAGRAM AND FORMULATION OF PIROXICAM NANOEMULSION GEL The pseudoternary phase plots were made by drawing double distilled water, oil mixture (1:2) and variable Smix ratios as 1:1, 2:1, 3:1, and 4:1 and as shown in figure (1). The shaded area of pseudoternary phase plot give the region of nanoemulsion, whereas the nonshade area offer the emulsion region. The Smix ratio (3:1) of pseudoternary phase plot has a greater nanoemulsifying region and from which a six formulas of nanoemulsion (NE1-NE7) were taken for characterization of nanoemulsions and to formulate nanoemulsion gel formulas (NF1-NF6). Figure (1): The pseudoternary phase diagram of double distilled water, oil mixture(1:2) and Smix (1:1, 2:1, 3:1, and 4:1). www.wjpps.com Vol 5, Issue 6, 2016. 313 Drais. World Journal of Pharmacy and Pharmaceutical Sciences CHARACTERIZATION OF THE FORMULATED PIROXICAM NANOEMULSION MEASUREMENT OF THE GLOBULE SIZE The results globule size range was NE1 (5-6.1nm), NE2 (5-7.29 nm), NE3 (7.2-9.2 nm), NE4 (13.1-15 nm), NE5 (18-23 nm) and NE6 (5-7 nm). The results indicate that all the formulas had droplets in the nanoscale size. Analysis of variance show significant differences between globule size and the concentration (%w/w) of Smix where (p<0.05). POLY DISPERSITY INDEX (PDI) MEASUREMENT PDI was from (0.01 to 0.019). The results of PDI indicate to that nanoemulsion formulations had a homogenous composition. EVALUATION PIROXICAM NANOEMULSION GEL PHYSICAL APPEARANCE The physical appearance of nanoemulsion gel formulas were yellowish in color,transparent and excellent homogenicity as shown in table (2). THE pH DETERMINATION The pH of all the nanoemulsion gels formulas was between 5.3 to 5.89 as in table (2) which are within the normal pH value of human skin. Table (2): The physical appearance, pH, percent of swelling index and percent of drug content in piroxicam nanoemulsion gel formulations. Formulation code NF1 NF2 NF3 NF4 NF5 NF6 color clarity uniformity yellow yellow yellow yellow yellow yellow clear clear clear clear clear clear excellent excellent excellent excellent excellent excellent Phase separation none none none none none none pH 5.3 5.55 5.89 5.66 5.44 5.51 % Swelling index 17 20 23 24 28 32 % Drug content 98.1 96.4 96.2 97.6 96.4 95.21 THE DRUG CONTENT MEASUREMENT The drug content was in a range of (95.21 – 98.1%) as shown in table (2). SWELLING INDEX ESTIMATION The outcome described in table (2). The result shows the effect of Smix on swelling index at constant gelling agent concentration. As the Smix concentration increase that lead to create more hydrophobic media that retard water molecule from enterance to formula therefore it www.wjpps.com Vol 5, Issue 6, 2016. 314 Drais. World Journal of Pharmacy and Pharmaceutical Sciences was found NF6 significantly higher swelling index due to it contain least Smix quantity and NF1 with higher Smix concentration has significantly lower swelling index (p<0.05). IN VITRO RELEASE MEASUREMENT The drug release profile in figure (2) indicates that the NF6 was significantly highest in dissolution rate while piroxicam gel formula has lower in dissolusion rate which was significantly lowest (P<0.05). The comparability drug release diagram of piroxicam nanoemulsion gel (NF1-NF6) and piroxicam gel show that the drug release was followed the descending order: NF6>NF5>NF4> NF3>NF2>NF1> piroxicam gel. The drug release profile from piroxicam nanoemulsion gel preparations (NF1-NF6) show the effect of Smix concentration at constant concentration of oil mixture and gelling agent,as the concentration Smix increase this lead decrease drug release this may be due to the high concentration of Smix increase hydrophobicity and retarding forces of formulations and resist dissolution media molecules passage through hydrophobic matrix and release the drug also the piroxicam molecules will have greater diffusional pathway to reach the dissolution media therefore it was found NF6 has greater release profile compare to other of piroxicam nanoemulsion gel formulations due to it has the least concentration of Smix that make less retarding effect for dissolution molecules and lower diffusional pathway to drug molecules to reach the dissolution media. also, the piroxicam gel release profile has lowest release rate compare to other piroxicam nanoemulsion gels this due to nanoemulsion found in nanoemulsion gels give many advantages in compare to gel such as nanosize droplets and improve solubility of piroxicam this will enhance passage of dissolution media to hydrophobic matrix and lower diffusional pathway for piroxicam molecules to reach the release media that make piroxicam nanoemulsion gels superior and highly attractive in compare to piroxicam gels.[6, 15] From in vitro release analysis, it was found that NF6 is a selected formula where it has lower globule size (5-7 nm ) as shown in figure (3) , low PDI (0.019), excellent physical appearance, the normal value of pH (5.51 ), a higher percent of swelling index (32), accepted percent of drug content (95.21%) and higher release rate makes it faster in relieving inflammatory conditions and enhance therapeutic efficacy. The selected formula (NF6) subject for further analysis of atomic force microscopy (AFM) study. www.wjpps.com Vol 5, Issue 6, 2016. 315 Drais. World Journal of Pharmacy and Pharmaceutical Sciences Figure (2): Represent drug release profile for piroxicam nanoemulsion gel and piroxicam gel formulations. Figure (3): The particle size distribution for NF6. ATOMIC FORCE MICROSCOY (AFM) ANALYSIS The result of AFM shows that the selected formula(NF6) has nano scale particle nearly spherical in shape and smooth surface globules, this mean the stability of optimized nanoemulsion gel (NF6) as shown in figure (4). www.wjpps.com Vol 5, Issue 6, 2016. 316 Drais. World Journal of Pharmacy and Pharmaceutical Sciences Figure (4): The AFM image of NF6 where scanning area is 5 μm * 5 μm. CONCLUSION 1. The aqueous phase titration method is a cheap and easy method that employed in the preparation of piroxicam nanoemulsion. 2. The piroxicam nanoemulsion gel (NF6) with its attractive physical characterstics and higher in vitro release rate will improve piroxicam solubility and bioavailability that lead to increase piroxicam therapeutic efficacy and enhance patient compliance. 3. The AFM study indicate that the optimized formula (NF6) with nanometer scale has good stability that makes it promising formula to local and systemic delivery of piroxicam. REFERENCES 1. Sweetman SC. Martindale the Complete Drug Reference. Chicago. the Pharmaceutical Press., 2009; 117-8. 2. Gutierrez JM, Gonzalez C, Maestro A, Sole I, Pey CM, Nolla J. Nano-emulsions: new applications and optimization of their preparation. Curr Opin Colloid Interface Sci, 2008; 13: 245–51. 3. Yilmaz E, Borchert HH. Effect of lipid-containing, positively charged nanoemulsions on skin hydration, elasticity and erythema — An in vivo study. Int J Pharm., 2006; 307: 232-8. 4. Dhawan Bhavna, Aggarwal Geeta, HarikumarSL. 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Formulation and evaluation of clotrimazole from pluronic F127 gels. Drug Discov Therap, 2010; 4: 33-43. 14. Karthikeyan S, Jeeva PA, Jerobin J, Mukherjee Amitava, Chandrasekaran N. Formulation and characterization of nanoemulsion coatings from Azadirachta indica. Int J ChemTech Res, 2012; 4: 1566-70. 15. Idrees M, Rahman N, Ahmad S, Ali M, Ahmad I. Enhance transdermal delivery of fl urbiprofen via microemulsions: Effects of different types of surfactants and cosurfactants. Daru., 2011; 19: 433-9. www.wjpps.com Vol 5, Issue 6, 2016. 318