Development and Optimization of Proniosomal Gel Containing Etodolac: In-vitro, Ex-vivo and In-vivo Evaluation

E-ISSN: 2340-9894 ISSN: 0004-2927 https://revistaseug.ugr.es/index.php/ars doi: 10.30827/ars.v62i3.17944 Artículos originales Development and Optimization of Proniosomal Gel Containing Etodolac: Invitro, Ex-vivo and In-vivo Evaluation Desarrollo y optimización del gel proniosomal que contiene etodolaco: evaluación in vitro, ex vivo e in vivo Moreshwar Patil1 orcid 0000-0002-8060-1687 Prashant Pandit2 orcid 0000-0003-1458-9381 Pavan Udavant3 orcid 0000-0001-6870-7852 Sandeep Sonawane4 orcid 0000-0002-4858-7651 Deepak Bhambere5 orcid 0000-0002-7880-9606 1,2,5Savitribai Phule Pune University, Pune, Department of Pharmaceutics, MET’s Institute of Pharmacy, Nashik 422 003, India. 3Savitribai Phule Pune University. Department of Pharmacology, MET’s Institute of Pharmacy, Nashik 422 003, Pune, India. 4Savitribai Phule Pune University Department of Pharmaceutical analysis, MET’s Institute of Pharmacy, Nashik 422 003, Pune, India. Correspondence Patil Moreshwar P. moreshwarpp[email protected]om Received: 11.01.2021 Accepted: 25.04.2021 Published: 21.06.2021 Funding This work has not been financially funded by any funding agency Conflict of interest The authors declare no conflict of interest. Ars Pharm. 2021;62(3):290-304 290 Resumen Introducción: Etodolac se usa en el tratamiento del dolor agudo y la inflamación. Tiene baja solubilidad debido a la alta hidrofobia y se informa que tras la administración oral muestra alteraciones gástricas. Esto fomenta el desarrollo de formulación tópica en lugar de oral. Método: En este trabajo utilizamos el método de separación de fase de coacervación para el desarrollo del sistema vesicular cargado con etodolaco mediante el uso de tensioactivos no iónicos, colesterol y lecitina de soja. El diseño central compuesto (rotativo) se utilizó para optimizar las concentraciones de lecitina de soja, surfactante y colesterol. Las formulaciones preparadas se caracterizaron por análisis de tamaño de vesículas, potencial zeta, eficiencia de atrapamiento, permeación in vitro, permeación ex vivo y estudio antiinflamatorio. Resultados: Etodolac quedó atrapado con éxito en todas las formulaciones que tenían una eficiencia en el intervalo de 74,36% a 90,85%, siendo mayor a 4 ° C que a temperatura ambiente. Cuando se hidrata con agua, los niosomas se producen espontaneamente el rango de 54 a 141 (por mm cúbico). Los resultados del estudio de difusión in vitro revelaron que el etodolaco se liberó en un rango de 71,86 a 97,16% durante un período de 24 horas. El tamaño medio de vesícula de la formulación optimizada se encontró en 211,9 nm con un PDI de 0,5. Las respuestas observadas, es decir,% de eficacia de encapsulación y liberación de fármaco, fueron 74,12 y 95,08 respectivamente. El potencial zeta fue de -19,4 mV y reveló la estabilidad de la formulación, que fue confirmada adicionalmente por la ausencia de cambios en el contenido del fármaco y la liberación del fármaco después de los estudios de estabilidad. El% de inhibición en el volumen de la pata fue del 40,52% y del 43,61% para la prueba y el gel proniosómico comercializado. Conclusión: la formulación de gel proniosomal fue estable y podría mejorar el suministro de etodolaco a la piel debido a la excelente capacidad de permeación del sistema vesicular. Palabras clave:administración de fármacos vesiculares; gel proniosómico; etodolaco; tensioactivos no iónicos; estudio antiinflamatorio. Abstract Introduction: Etodolac is used in the treatment of acute pain and inflammation. It has low solubility because of high hydrophobicity and it is reported that upon oral administration shows gastric disturbances. This encourages the development of topical vesicular formulation. Method: In this work we used coacervation-phase separation method for the development of etodolac loaded vesicular system by using non-ionic surfactants, cholesterol and soya lecithin. Central composite design (rotatble) was used to optimize the concentrations of soy lecithin, surfactant and cholesterol. The prepared formulations were characterized by number of vesicles formed, vesicle size, zeta potential, entrapment efficiency, in-vitro permeation, ex-vivo permeation and anti-inflammatory study. Results: Etodolac was successfully entrapped in all formulations having efficiency in the range of 74.36% to 90.85%, which was more at 4 °C than room temperature. When hydrated with water; niosome in the range of 54 to 141 (per cubic mm) were spontaneously produced. The results of in-vitro diffusion study revealed that etodolac was released in the range of 71.86 to 97.16% over a period of 24 hrs. The average vesicle size of optimized formulation was found 211.9 nm with PDI of 0.5. The observed responses i.e. % encapsulation efficiency and drug release were 74.12 and 95.08 respectively. The zeta potential was -19.4mV revealed the stability of formulation which was further confirmed by no changes in drug content and drug release after stability studies. The % inhibition in paw volume was 40.52% and 43.61% for test and marketed proniosomal gel. Conclusion: Proniosomal gel formulation was stable and could enhance skin delivery of etodolac because of excellent permeation capability of vesicular system. Keywords:Vesicular drug delivery; proniosomal gel; etodolac; non-ionic surfactants; antiinflammatory study. Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 291 Highlights • Topically applied drugs can be delivered as proniosomal vesicular system. • Vesicular system can be used to increase the penetrability of the drug. • Span, soy lecithin and cholesterol can be used to develop stable formulation. • Statistical technique like central composite design would be helpful in formulation optimization. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently prescribed for arthritis, low back pain and some joint diseases(1) . The well reported mechanism of action is reversible inhibition of cyclooxygenase enzyme (COX) and decrease the synthesis of prostaglandins. However, due to inhibition of prostaglandins (PGs) which protects the gastric mucosa, they show side effects including dyspepsia to peptic ulcer and gastrointestinal haemorrhage. NSAIDs are acidic in nature they may produce local irritation and lesions on the gastrointestinal mucosa. Hence, some of the NSAIDs are administered percutaneously and transdermally to achieve local or systemic effect as an alternative to oral and parenteral administration(2). However, the barrier layer i.e. stratum corneum prevents the penetration of the drugs to lower layers of the skin and/or to enter systemic circulation. In this context, the formulation plays a key role in the penetration and absorption of the active ingredient. 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. It has poor water solubility (75 µg/ml) because of high hydrophobicity(3). It causes gastric irritation, constipation, diarrhoea, vomiting, headache dizziness, sore throat and blurred vision(4) and may create limitation in formulation of oral dosage forms. By considering the drawbacks of oral delivery of etodolac; it would beneficial to give it through topical route using novel vesicular drug delivery system. It was reflected from the literature that niosomes; a vesicular system could be beneficial for topical and transdermal delivery because they act as a reservoir of drug for a prolonged period of time and enhance skin penetration(5,6) and thus increases bioavailability. However the niosomes are also has instability issues like fusion, aggregation, sedimentation and leakage of entrapped drug. These mentioned problems can be avoided by using non ionic surfactant formulation which is anhydrous, free flowing i.e. proniosomes and can form vesicles after hydration (niosomes). Proniosomes can entrap both hydrophilic and lipophilic drugs. Proniosomal gel is beneficial to reduce drugs associated side effects, increase effectiveness and make the formulation physically stable. In this context we had considered development of vesicular topical formulation with optimal quality in short period of time with less number of experimental trials using varying proportion of soy lecithin, cholesterol and non-ionic surfactant(7). Hence, two level three factor rotatable central composite design (CCD) was used study the effect of amount of soy lecithin, non ionic surfactant and cholesterol on entrapment efficiency (%EE) and % drug release. Materials and Methods Materials Etodolac was received as a gift sample from Swiss Garnier Life Sciences, Una, Himachal Pradesh, India. Lipoid S 100 and Phospholipon 80H were obtained from Ludwings Shafen, Germany. Span (20, 40 and 60) was purchased from S. D. Fine Chem. Ltd., Mumbai, India. Cholesterol was obtained from Thomas Baker Ltd., Mumbai, India. All other chemicals used in this study were of analytical grade. Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 292 Methods Preformulation study To eliminate the possibility of interaction between etodolac and cholesterol, lecithin and Span, compatibility study was carried out by storing the physical mixture (1:1) in the stability chamber at 40 °C/75% RH for 1 month. At the end of the study, FT-IR spectrums of etodolac alone and binary mixture were recorded and studied. Prior incorporation of etodolac, trial formulation batches containing varying proportions of Span (20, 40 and 60): lecithin (Lipoid S 75, Phospholipon 80 H, Lipoid S 100) in ratio of 0.5:1, 1:1, 1:0.5 and 1:0.75 were developed. These preparations were observed for homogeneity, consistency, clogging and sudden change in viscosity. Development of proniosomal gel Etodolac proniosomes were prepared by modified coacervation phase separation method reported by Thakur et. al(8). Briefly, etodolac (5% w/w) and varying concentrations of Span 60, Lipoid S 100 and cholesterol were mixed with 2.5 ml of absolute ethanol in a wide mouth glass tube. The ingredients were mixed together; open end of the glass tube was covered and warmed on a water bath at 60-70 ºC for about 5 min. Phosphate buffer saline (pH 7.4) was added and warmed on a water bath for ≈2 minutes to form dispersion. The mixture was allowed to cool to room temperature until the dispersion was converted to proniosomes having semisolid consistency and stored in dark until further use. Optimization by design of experiment A full central composite design (rotatable) was used in this study. Design matrix is given in Table 1. The amount of Span 60 (A), Lipoid S 100 (B) and cholesterol (C) were taken as independent variables, while % entrapment efficiency (Y1) and % drug release (Y2) were taken as dependent variables. The factors were studied at 2 levels (−1, +1) indicating low and high respectively. The design consisted of three groups of design points, including two level three factor factorial design points, axial or star points and center points. Total 20 experiments consisting of 8 factorial points, 6 axial points, and 6 replicated central points were performed according to the central composite design matrix generated by Design Expert V. 11 Software (Table 2) (Stat-Ease, Minneapolis, USA). Table 1: Two level three factor central composite design Factor Low level High level -α +α Experimental domain (Coded values) Span 60 (mg) -1 +1 -1.682 +1.682 Lipoid S 100 (mg) -1 +1 -1.682 +1.682 Cholesterol (mg) -1 +1 -1.682 +1.682 Experimental domain (Actual values) Span 60 (mg) 1200 1800 995.46 2004.5 Lipoid S 100 (mg) 500 1000 329.55 1170 Cholesterol (mg) 50 100 32.96 117.04 Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 293 Table 2: Experimental design batches of proniosomes Experiment Span 60(mg) Lipoid S 100 (mg) Cholesterol (mg) Ethanol(ml) Saline sol. (ml) Factorial designs CD 1 1200 500 50 2.5 1.8 CD 2 1800 500 50 2.5 1.8 CD 3 1200 1000 50 2.5 1.8 CD 4 1800 1000 50 2.5 1.8 CD 5 1200 500 100 2.5 1.8 CD 6 1800 500 100 2.5 1.8 CD 7 1200 1000 100 2.5 1.8 CD 8 1800 1000 100 2.5 1.8 Star design CD 9 995.46 750 75 2.5 1.8 CD 10 2004.5 750 75 2.5 1.8 CD 11 1500 329.55 75 2.5 1.8 CD 12 1500 1170.45 75 2.5 1.8 CD 13 1500 750 32.96 2.5 1.8 CD 14 1500 750 117.04 2.5 1.8 Center points CD 15 1500 750 75 2.5 1.8 CD 16 1500 750 75 2.5 1.8 CD 17 1500 750 75 2.5 1.8 CD 18 1500 750 75 2.5 1.8 CD 19 1500 750 75 2.5 1.8 CD 20 1500 750 75 2.5 1.8 Evaluation of proniosomal gel Determination of pH and viscosity The pH of all formulations was determined by using pH meter at ambient temperature. The pH meter was calibrated before each use. About 1g of gel was dissolved in 25ml of distilled water and pH was measured by immersing the electrode. The viscosities of different proniosomal gel formulation were determined at 25°C using a Brookfield viscometer. The formulation (more than 5g) was placed in a beaker and was allowed to equilibrate for 5 min before measuring the dial reading using a spindle no S-64 at 20 rpm(6,9). % Encapsulation efficiency (%EE) Approximately 0.2 g of proniosomal gel was dispersed in 10 ml of phosphate buffer (pH 7.4). The dispersion was sonicated, centrifuged at 18,000 rpm at -20°Ċ for 30 min. (C-24 BL, Remi lab) to separate Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 294 unentrapped drug. The supernatant was removed and quantified by an UV spectrophotometer at 278 nm(10). The % encapsulation was calculated by the following equation: Where, Ct is the concentration of total etodolac; Cf is the concentration of free etodolac. Vesicle size and zeta potential Vesicle size, size distribution and zeta potential were determined by Malvern Zetasizer in order to ascertain the stability as well as the reproducibility of the method. About 1g of gel was dissolved in 20 ml double distilled water and agitated to get homogeneous dispersion. Mean globule diameter and zeta potential was recorded(11). Rate of Spontaneity This test helps to determine the numbers of niosomes formed spontaneously after hydration in 15-20 min. Approximately 100 or 200 mg of proniosomal gel was transferred to glass bottle and spread uniformly around walls. About 20ml of saline solution was added along the walls and was left aside for 20 min. then a drop was withdrawn and place on Neubauers chamber to count the number of vesicles(12). In-vitro and Ex-vivo drug release In-vitro release study was performed by using diffusion cell apparatus and egg membrane. Egg membrane was separated by putting the egg shell in mixture of water and hydrochloric acid in which the shell is dissolved to calcium chloride with effervescence of carbon dioxide. Before initiation of study; the egg membrane was soaked in phosphate buffer for 24 hours. About 1g of proniosomal gel was placed in the donor compartment tube and spread over the egg membrane which was clamped at the end of the tube. The acceptor compartment consists of phosphate buffer (pH 7.4). The entire surface of the cell was in contact with the medium present in the receptor compartment which was agitated by magnetic stirrer at rpm 600 and temperature 32±1°Ċ. A sample was removed at specific time interval for up to 24 h with same amount replaced to maintain sink condition. The sample was analysed at 278 nm against blank using UV spectrophotometer. For ex-vivo study; the egg membrane was replaced by excised skin of abdominal region of rat. The stratum corneum of the skin faced the drug donor compartment whereas dermis faced the receiver compartment(13-16). Scanning electron microscopy The surface morphology of the formulation was determined by placing the drop of niosome dispersion on carbon coated grid, vacuum dried and observed microscopically at an accelerating voltage of 10 Kv using scanning electron microscope (JEOL/JEM 2100, Japan). Anti-inflammatory study The study protocol was approved by the Animal Ethical Committee of MET’S Institute of Pharmacy (CPCSEA Reg. no-1344/ac/10/CPCSEA/IAEC) and it was conducted as per the guidelines. Fifteen albino mice were selected. They were divided into 3 groups (control, standard and test) each containing 5 animals and kept fasted overnight. About 0.1ml of 1 % (w/v) carrageenan was injected on the left hind paw to induce edema. Control group received carrageenan (1% w/v) only while standard and test were treated with marketed and developed proniosomal gel in addition to carrageenan. The paw thickness was measured using vernier calliper at the time interval of 0, 30, 60, 120 and 180 minute. The % inhibition of diameter paw edema by proniosomal gel and marketed gel treated group compared with carrageenan control group(17). From the mean edema volume, the percentage inhibition of the edema Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 295 was calculated between the treated and control groups. Before anti-inflamatory activity skin irritation study was conducted to find out the allergic potential of formulation. Where, Vc = Paw volume of control Vt = Paw volume of test Vs = Paw volume of standard Results and Discussion Preformulation study The FT-IR spectrums of etodolac, etodolac with Span 60, Lipoid S-100 and cholesterol are shown in Figure 1. The principle peaks of the etodolac i.e. –NH stretching at 3342.64 cm-1, -CH stretching in alkane at 2970.38 cm-1 and –CO stretching in ketone at 1745 cm-1, –CO stretching in aromatic at 1261 cm-1 were present in all physical mixtures. There was neither deletion of existing peak nor addition of new peak in the spectrum revealed the compatible nature of the excipients with etodolac. Hence; by using these materials stable formulations were developed. Figure 1. FTIR spectrums for prediction of incompatibility: etodolac and its binary mixture with Span 60, Lipoid S 100 and cholesterol. Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 296 Preliminary studies have demonstrated that formulations of Span 20 and 40 with Lipoid S 100 and Phospholipon 80 H were transparent; yellow and semisolid. Gels of Span 40 had shown clogging with change in viscosity after 1 week of storage. In case of Span 20, formulations were stable but less amount of etodolac was incorporated. Hence; it would be beneficial to use Span 60 as it had shown high entrapment efficiency in addition to consistency. Similarly niosome vesicles prepared with Lipoid S 100 were observed good and denser than prepared with Phospholipon 80 H. Data analysis by central composite design Effect of composition variable on %EE Proniosomes are made of lipidic materials and considered as it has ability to entrap drug. The response (Y1) i.e. %EE was ranged from 74.36±0.22 to 90.85±0.26 (Table 3). ANOVA test revealed that the response surface quadratic model was significant. The polynomial equation for encapsulation efficiency is %EE=+81.42-1.58*A-2.35*B+1.75*C-2.19*A*B-2.30*A*C+0.96*B*C-0.88A2-0.13B2+0.90C2 It was observed from the equation and perturbation plot (Figure 2) that the amount of Span 60 and Lipoid S 100 had negative effect on encapsulation while amount of cholesterol had positive effect indicating that increase in concentration of cholesterol encapsulate more amount of drug. It may be due to the fact that cholesterol forms hydrogen bond with non ionic surfactant and impart stability to niosomes and hence, more amount of drug is encapsulated(18). It is reported that Span 60 has HLB value of 4.7 and decreased HLB value is also responsible for decreases encapsulation efficiency than that of surfactant with high HLB(19). Table 3: Results of responses measured for 20 CCD batches Experiment Y1 (% EE) (Mean±SD) Y2 (% CDR) (Mean±SD) CD 1 80.85±0.19 74.8±0.94 CD 2 89.31±0.32 84.17±0.65 CD 3 80.18±0.31 71.86±0.43 CD 4 76.89±0.12 95.4±0.64 CD 5 84.75±0.24 90.53±0.84 CD 6 81.03±0.34 90.9±0.89 CD 7 84.94±0.16 85.47±0.51 CD 8 75.46±0.18 96.06±0.14 CD 9 82.42±0.24 85.01±0.72 CD 10 74.36±0.22 97.16±0.69 CD 11 84.59±0.15 90.9±0.5 CD 12 76.48±0.28 95.63±0.94 CD 13 76.01±0.37 82.1±0.63 CD 14 90.85±0.26 80±0.66 CD 15 81.2±0.23 87.3±0.35 CD 16 81.37±0.27 86.76±0.83 CD 17 81.76±0.33 85.3±0.45 CD 18 81.23±0.28 84.15±0.81 CD 19 81.66±0.35 86.13±0.34 CD 20 81.46±0.16 87.46±0.81 Optimized 83.74±0.72 86.62±0.85 Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 297 Figure 2. Perturbation plot for % encapsulation efficiency (EE). Effect of composition variable on % drug release The percentage cumulative drug release was observed in the range of 71.86±0.43 to 97.16±0.69 (Table 3). Following is the polynomial equation for %CDR: % CDR= +86.26+4.71*A+1.20*B+2.43*C+3.05*A*B-2.74*A*C-1.02*B*C+1.21*A2+1.99*B2-2.33*C2 The component of formulation; Span 60, Lipoid S 100 and cholesterol had positive coefficients. The perturbation plot (Figure 3) reveled that curvature of factor A i.e. Span 60 demonstrated significant proportional effect on drug release as compare to Lipoid S 100 and cholesterol. It was observed from the release profile that the vesicular system prolonged the release of etodolac for 24 hrs. The release studies have demonstrated presence of some amount of drug may bind to surface because of that initial burst release of etodolac followed by slow release was observed. The prolonged release for longer period of time could improve patient compliance as it reduces application frequency(20). Ars Pharm. 2021;62(3):290-304 Patil M, Pandit P, Udavant P, et al. 298