Gokaraju Rangaraju College of Pharmacy, India
Trapti Saxena is working as Senior Assistant Profesor in Andhra Pradesh 1st NBA Accredited Pharmacy College. She has supervised 5 project students for post graduate research work in Novel Drug Delivery Systems. She has 2 publications to her account. Apart from 9 years of academic experience, she has 5 years of research experience.
Itraconazole, a triazole antifungal drug, belongs to BCS class II which suffers from poor solubility and finally results decreased bioavailability. A solid self-nanoemulsifying drug delivery system (SNEDDS) was developed for oral bioavailability enhancement of itraconazole. The liquid SNEDDS of itraconazole was prepared using NMP (oil), Labrasol (surfactant) and Transcutol (co-surfactant). Most of the liquid formulations exhibited more than 85% of drug release in 60 min. The nano-size was obtained for optimized liquid SNEDDS formulation (T26). The pharmacokinetic study in rats was performed. SNEDDS formulation has significantly increased the absorption rate when compared to the pure drug. The liquid SNEDDS formulation, T26 was adsorbed on solid carrier Neucilin in different L-SNEDDS:carrier ratios. Among 9 formulations, formulation ratios 2:1 exhibited passable powder flow property. The in vitro drug release was 7 % in 60 mins for pure drug, while from solid SNEDDS formulation N1 was 87 %. The particle size was 138 nm and zeta potential value was within the range (-14.3) for N1. DSC thermogram and SEM micrographs indicated that the drug is solubilized. Stability studies indicated a miniscule variation in the values (emulsification time, drug content and % release of itraconazole) during three months of their storage. This work provides an overview of the SNEDDS of Itraconazole as a promising alternative to improve oral absorption. Key words: Itraconazole, SNEDDS, pseudoternary phase diagrams, NMP, Labrasol. INTRODUCTION Oral route has always been the favorite route of drug administration in many diseases and till today it is the first way investigated in the development of new dosage forms. The oral delivery of lipophilic drugs presents a major challenge because of the low aqueous solubility. Approximately one third of the drugs emerging from drug discovery programs are poorly water soluble, presenting the pharmaceutical scientist with several problems when developing formulations for such active pharmaceutical ingredients. SNEDDS are defined as isotropic mixture of oils, surfactants and co-surfactants that distribute readily in the GI tract. These typically produce nanoemulsions in the size range of 20-200 nm and provide the drug in dissolved state on account of a large interfacial area for the drug at the absorption site (5). When compared with emulsions which are sensitive and metastable dispersed forms, nanoemulsions are physically stable formulations that are easy to manufacture. Thus, these systems may offer an improvement in the rate and extent of absorption and result in more reproducible blood time profiles (1). Itraconazole (ITZ), a triazole antifungal agent, shown to have a broad spectrum of activity against a variety of pathogens, which are the major cause of opportunistic infection in human immunodeficiency virus (HIV) infected patients. It is a weak basic drug (pKa = 3.7), which is virtually ionized at only low pH, and however possessing extremely low water solubility. The log PC of itraconazole is 5.66 in n-octanol-aqueous buffer solution, pH 8.1. It indicated a very high lipophilicity. According to the BCS, it is in class II, suggesting poor aqueous solubility. Therefore, itraconazole is suitable for studying the utility of nanoemulsions containing poorly water-soluble drug. SMEDDS can exist in either liquid or solid dosage form. SMEDDS are usually, limited to liquid dosage forms but they have disadvantages like high production costs, low stability and portability, low drug loading and few choices of dosage forms. More importantly, the large quantity (30–60%) of surfactants in the formulations can induce gastrointestinal (GI) irritation. To solve these problems, Solid SMEDDS (S-SMEDDS) have been investigated, as an alternative approach. Such system requires the solidification of liquid self-microemulsifying (SE) ingredients into powders/nano-particles to incorporate into various solid dosage forms. Thus, S-SMEDDS combine the advantages of SMEDDS (i.e. enhanced solubility and bioavailability) with those of solid dosage forms (e.g. low production cost, convenience of process control, high stability and reproducibility, better patient compliance) (2). MATERIALS AND METHODS Materials Itraconazole, NMP (N-methyl-2-pyrrolidone), Labrasol, Cremophor RH-40, Vitamin E TPGS, were obtained as a gift samples from Mylan Laboratories Private Limited (Hyderabad). Neucilin and Syloid were obtained as gift samples from Dr Reddy’s Laboratories, Hyderabad. Tween 80, Transcutol P, PEG 400, propylene glycol, methanol, concentrated hydrochloric acid were purchased from SD Fine Chemicals (Mumbai). Preparation of Liquid SNEDDS A series of self-emulsifying systems were prepared with varying concentrations of oil, surfactant, and co-surfactant. In all the formulations, a fixed amount of itraconazole was added. The oil, surfactant and co-surfactant were blended in a glass vial and this blend was kept in a water bath at 40-50 °C. Itraconazole and 35% hydrochloric acid (minimum required amount) were added with constant stirring until a clear solution was obtained. The mixture was stored at room temperature until used. In vitro itraconazole release studies from liquid SNEDDS The In vitro drug release of Itraconazole was performed by a conventional method using dissolution apparatus USP II. A hard gelatin capsule size ‘0’ filled with pre-concentrate (equivalent to 50 mg itraconazole) and placed into 900 ml of 0.1N hydrochloric acid solution. The samples were filtered and analyzed using UV spectrophotometer at 255 nm. Similar studies were carried out for pure itraconazole. Droplet size analysis and Zeta potential measurement The average droplet size and Zeta potential of SNEDDS were measured by photon correlation spectroscopy using a Malvern Zetasizer (Nano ZS 90, Malvern instrument ltd., U.K.). The formulation (0.1 ml) was dispersed into 100 ml of water. Then 1ml aliquot was withdrawn and added into sample cell. The measurements were performed at 25 °C at a fixed angle of 90° (18). In vivo studies All experiments were performed according to animal ethical committee. Male Albino rats, weighing approximately 250 g, were obtained from animal house. The animals were fasted for 12 h before drug administration but were allowed free access to water. The animals were divided into two groups (six animal each) and each animal received one of the following dosage form: Itraconazole SNEDDS and Itraconazole suspension containing drug equivalent to 15 mg/kg of body weight. The formulations were administered by the oral route with a gastric catheter. Blood sample were withdrawn at designated time intervals through retro orbital plexux. The concentration of itraconazole in rat plasma was determined by HPLC analysis (3). Preparation of solid smedds of drugs The optimized liquid SMEDDS formulation of drug was solidified by adsorption method. The solid carriers, having high adsorbancy and surface area, such as Neusilin US2, Syloid and Aerosil 200 were used at various carriers to SMEDDS ratios (1:2, 1:1, 2:1). The SMEDDS formulations were added drop wise over the solid adsorbent contained in a porcelain dish. After each addition, the mixture was homogenized using glass rod to ensure uniform distribution of the formulation. Resultant mass was passed through sieve no. 80 and stored in a dessicator until further use (4). Evaluation of solid SNEDDS Preformulation properties: Angle of repose, bulk density, tapped density, Hausner’s ratio and Carr’s index were determined as per standard pharmacopoeial methods In vitro drug release studies from solid SMEDDS: The in vitro dissolution study of solid SMEDDS and plain drugs was carried out by using USP Type II dissolution apparatus. The solid SMEDDS, containing itraconazole equivalent to 100 mg were filled in ‘0’ size capsules and placed in the flask of the dissolution apparatus. An aliquot were withdrawn at predetermined intervals and analyzed. Differential scanning calorimetry (DSC): The powder samples was hermetically kept in the aluminium pan and heated at constant rate 10 °C/min, over a temperature range of 0 °C to 450 °C. Inert atmosphere was maintained by purging nitrogen at the flow rate of 60 ml/min. Scanning electron microscopy (SEM): Samples were fixed on a brass stub using double sided adhesive tape and were made electrically conductive by coating with a thin layer of gold and SEM images were recorded at 30 kV accelerating voltage. RESULTS AND DISCUSSION Preparation of itraconazole loaded liquid SNEDDS formulations Preliminary studies were performed for the solubility of itraconazole. NMP as oil and Labrasol as surfactant had high solubility. Further, Transcutol P and PEG 400 can be considered as co-surfactants. Through ternary phase diagrams, the nanoemulsion region was identified. All forty one formulations exhibited emulsification time as less than 20 seconds. Phase separation, drug precipitation, thermodynamic stability and robustness to dilution studies were satisfactory. To liquid SNEDDS 100 mg of itraconazole was added. In vitro itraconazole release studies from liquid SNEDDS The in vitro release profile showed a significant increased rate of dissolution, when compared with the pure drug, in case of all 41 formulations. The order of kinetics of itraconazole release from liquid SNEEDS formulations was found to be first order. The mechanism of itraconazole release satisfied Higuchi’s equation, diffusion rate controlled. Results indicated an instantaneous increase in the itraconazole release from SNEDDS, similar to burst effect type, more than 40% in 10 min. This suggested the importance of nanosize, though the formulation is lipid based. Droplet size analysis and zeta potential measurement Droplet size and zeta potentials measurement were carried out for selected 6 formulations. Droplet size ranged from 80 to 613 nm and the zeta-potential results indicated the range -6.23 to -30.6 mV. Based on particle size (80 nm), zeta potential (-26.7 mV) and in vitro drug release (98.68±2.19) and other evaluation tests, formulation T26 (20% NMP, 68.57 % Labrasol and 11.4% Transcutol P) was considered superior and selected for solidification. In vivo studies The concentration of itraconazole in the plasma samples were determined by HPLC. The peak areas were used to calculate the plasma concentrations of itraconazole and the SNEDDS formulation.. The perusal to Fig 4-1 indicated that the pure drug has a very low absorption, may be on account of its low solubility. SNEEDS formulation (T26) exhibited enhanced rate of absorption. The absorption data indicated the ascending absorption phase. Hence the data were processed under linear regression equation. The slopes are 0.1146 and 0.0065 for SNEDDS and itraconazole alone, respectively. The rate was approximately doubled. In vivo studies indicated that about 46% increase in plasma concentration from SNEDDS formulation, compared to the itraconazole. Figure 4-1: Comparison of plasma concentrations-time profiles of pure drug and itraconazole SNEDDS formulation in rats Characterization of solid self-microemulsifying powder Solid SNEDDS were evaluated for powder flow properties, reconstitution properties and subjected for solid state characterization. Preformulation properties The solid SNEDDS prepared in this work were evaluated for different properties such as angle of repose, bulk density, tapped density, carr’s index and hausner’s ratio. From the results it was observed that 1:1 and 2:1 ratios (L-SNEDDS:solid carrier) showed uniform distribution and good flow properties. In-vitro drug release studies It was concluded that the release percentage of itraconazole from solid SNEDDS formulations was significantly higher than that of pure drug. SNEDDS prepared using Neusilin US2 (N1) showed more release than that prepared with Aerosil 200 (A2) and Syloid (S2). The itraconazole (alone) release was 7 % in 60 mins, while from N1 was 87 %.Formulation, N1 showed highest drug release of 93.03 % in 120 min. The drug release from solid SNEDDS was compared with optimized liquid SNEDDS formulation and pure drug (Figure 4-2). In liquid SNEDDS, release was higher than that of S-SNEDDS. This is understandable because solid SNEDDS require longer emulsification time. Figure 4-2: Comparative release of itraconazole from solid SNEDDS, liquid SNEDDS formulation and itraconazole pure form Particle size and zeta potential of solid SNEDDS: The particle size is satisfactory as the size is in nanometer. The zeta potential value was satisfactory as it was within the range. Thus the solid SNEDDS (N1) is stable with the desired size. Solid State Characterization of Solid SNEDDS: The DSC curves of pure itraconazole showed a sharp melting endothermic peak at about 166 °C, indicating its crystalline nature. However, the endothermic peak of itraconazole was absent in the SNEDDS formulation prepared with neusilin US2. Thus it can be confirmed that the drug has got solubilized into the excipients of the SNEDDS. Morphological Analysis of solid SNEDDS The SEM pictures of Neusilin US2 (carrier) and S-SNEDDS (formulation N1) are shown in Figures 4-3 (A & B). According to SEM images, the carrier (Neusilin US2) was appeared as porous particles with a rough surface. However, the solid SNEDDS appeared as smooth-surfaced particles, indicatingthat the liquid SNEDDS is adsorbed onto the surface of Neusilin US2 particles. A B Figure 4-3: Scanning electronic micrograph of A- Neusilin US2, B- itraconazole solid SNEDDS formulation (N1) Stability studies The stability profile of solid SNEDDS was evaluated in terms of emulsification time, drug content and % cumulative release of itraconazole (in 120 mins). The data shown miniscule variation in the values during three months of their storage at the stability conditions of 40 ± 2°C/65 % ±5 % RH. This confirms that the optimized formulation is stable throughout the period. Conclusions In this study, solid SNEDDS formulation of itraconazole was prepared for direct filling in hard gelatin capsules for oral administration and evaluated for their in vitro and in vivo behavior. The optimized formulation exhibited faster release profiles with a rapid rate of emulsification. The resultant emulsion was negatively charged, with a small mean size and a narrow particle size distribution. The optimized SNEDDS formulation of itraconazole showed a 46% increase in the oral absorption, compared with the pure itraconazole. Thus, SNEDDS can be regarded as a commercially feasible alternative to the current itraconazole formulations. Acknowledgements The authors want to acknowledge Mylan Laboratories Private Limited, Hyderabad for providing the gift samples of Itraconazole, NMP, Labrasol and Transcutol P and also for providing facility and assistance during droplet size analysis and zeta potential measurement. REFERENCES 1. Gupta RN, Gupta R, Rathore GS. Enhancement of Oral Bioavailability of Lipophilic Drugs from Self-microemulsifying Drug Delivery System (SMEDDS). Int J Drug Dev & Res 2009;1(1):10-18. 2. Paramita D, Sabyasachi M, Somasree R, Biswanath S, Sen K. Self- emulsification of poorly soluble and highly permeable drugs: An overview. International Journal of Pharma Recent Research 2009;1(1):67-72. 3. Tang B, Cheng G, Jian-Chun Gu, Cai-Hang Xu. 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