Development of Repaglinide Loaded Solid Lipid ... - IngentaConnect

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Dec 2, 2009 - Abstract: Repaglinide solid lipid nanoparticles (RG-SLN) were fabricated using stearic acid as lipid. Pluronic F68 (PL-. F68) and soya lecithin ...
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Current Drug Delivery, 2010, 7, 44-50

Development of Repaglinide Loaded Solid Lipid Nanocarrier: Selection of Fabrication Method M.K. Rawat1, A.Jain1, A. Mishra1, M.S. Muthu2 and S. Singh1,* Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi – 221005, India; Department of Pharmacology, Institute of Medical Science, Banaras Hindu University, Varanasi – 221005, India Abstract: Repaglinide solid lipid nanoparticles (RG-SLN) were fabricated using stearic acid as lipid. Pluronic F68 (PLF68) and soya lecithin were used as a stabilizer. SLNs were prepared by modified solvent injection and ultrasonication methods. SLNs prepared with modified solvent injection method have larger particle size (360±2.5nm) than prepared with ultrasonication method (281±5.3nm). The zeta potential of the prepared formulations by these two methods varied from 23.10 ±1.23 to -26.01 ±0.89 mV. The maximum entrapment efficiency (62.14 ±1.29%) was obtained in modified solvent injection method. The total drug content was nearly same (98%) in both the methods. In vitro release studies were performed in phosphate buffer (pH 6.8) with 0.5% sodium lauryl sulphate (SLS) using dialysis bag diffusion technique. The cumulative drug release was 30% and 50% within 2 hrs in modified solvent injection and ultrasonication method, respectively. This indicates that RG-SLN prepared from modified injection method released the drug more slowly than SLNs prepared with ultrasonication method. Differential scanning calorimetry indicates that repaglinide (RG) entrapped in the solid lipid nanoparticles (SLN) exist in an amorphous or molecular state. Repaglinide loaded solid lipid nanoparticles prepared with both methods were of spherical shape as observed by transmission electron microscopy (TEM). These results suggest that modified solvent injection method is more suitable for preparation of repaglinide SLNs using stearic acid.

Keywords: Modified solvent injection technique, ultrasonication, oral route, bioavaiability, colloidal dispersion. 1. INTRODUCTION Among the various routes of drug delivery, oral route is the most preferred to the patient and the clinician alike, although several factors like pH of GIT, residence time and solubility can affect the therapeutic activity of a drug given via the oral route. Approximately, 40% of drug candidates have poor water solubility and the oral delivery of such drugs is frequently associated with implications of low bioavailability, hepatic first pass metabolism, enzymatic degradation, high intra- and inter subject variability, and lack of dose proportionality [1]. Nowadays, the interest of solid lipid nanoparticles (SLN) to improve the oral bioavailability of poorly water soluble drugs are well known and documented with various drugs such as halofantrine [2], Clozapine [3], cyclosporine [4], quercetin [5]. SLN enhances lymphatic transport of the drugs, reduces the hepatic first pass metabolism and improves bioavailability, because intestinal lymph vessels drain directly into the thoracic duct, further in to the venous blood, thus by passing the portal circulation [6]. The lymphatic system facilitates the absorption of long chain fatty acids via chylomicron formation. The peroral bioavailability of various poorly soluble drugs had been improved by incorporating them in SLN. Demirel et al. [7] prepared piribidil SLN and administered them orally to rabbits. Bioavailability of piribidil was improved more than two*Address correspondence to this author at the Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi – 221005; India; Tel: + 91-542-2315871; Fax: + 91-542-2368428; E-mail: [email protected]; [email protected] 1567-2018/10 $55.00+.00

folds compared with pure piribidil when administered in SLN. Intraduodenal administration of tobramycin SLN to rats showed improved bioavailability compared with pure tobramycin solution [8]. The relative bioavailability of nitrendipine solid lipid nanoparticles was nearly 4 times more compared to nitrendipine control suspension, following oral administration to wistar rats [9]. Yang et al. [10] also found the bioavailability enhancement of praziquental loaded solid lipid nanoparticles after oral administration. Solid lipid nanoparticle has clear advantage of biocompatibility over polymeric nanoparticles as lipid matrix is made from physiological lipids which decrease the danger of acute and chronic toxicity [1]. Repaglinide (RG) belong to a new class of oral hypoglycemic agents known as the meglitinide analogues. It is approved by FDA in 1998 as a type II antidiabetic drug. It is practically insoluble in water and having poor bioavailability (50%) because of extensive hepatic first pass metabolism resulting in the formation of inactive metabolites. RG is rapidly cleared from the blood-stream with a terminal elimination half-life (t1/2) of 1 hour [10]. Studies demonstrate that multiple daily doses of RG are more efficient than sulphonylureas on improving glucose and mixed meal induced insulin secretion and has a more beneficial effect in reducing cardiovascular risk factors mainly through an antioxidant role [11]. RG cause less hypoglyceamia and weight gain than sulphonylureas [12]. In spite of having number of beneficial effects, RG is less preferred drug in comparison to other antidiabetic drugs because of its short half life and low bioavailability.

© 2010 Bentham Science Publishers Ltd.

Development of Repaglinide Loaded Solid Lipid Nanocarrier

Recently, some work on the use of calcium silicate based floating microsphere as a carrier for RG has been published [13]. It is reported in literature that calcium silicate is toxic (hemolytic activity) to human being [14]. Therefore, still there is a need for safe and effective drug delivery carrier that can improve the oral bioavailability of RG. It is already established that solid lipid nanoparticles are biocompatible and improve the oral bioavailability of poorly water soluble drug [1, 15]. Porter and Charman et al. [5] have previously suggested that a log partition coefficient value in excess of 5 is a minimum requirement for appreciable lymphatic transport. RG has 5.9 logP [16] which confirmed that RG is suitable molecule for lymphatic transport. In order to choose a better carrier material, an endogenous long chain saturated fatty acid (stearic acid) was studied in this investigation. The stearic acid being the main component of fat, so it was expected to have better biocompatibility and lower toxicity than the polymers. Several approaches on preparation [17, 18] and stability [19, 20] aspects have been reported about solid lipid nanoparticles made from stearic acid. Pluronic F68 (PL-F68) was used as stabilizer in the preparation. Apart from the excipients used for the preparation of solid lipid nanoparticles, the method of preparation greatly influences the particle size, entrapment efficiency, in vitro drug release and physical stability of SLN. The standard production method for the preparation of solid lipid nanoparticles is high pressure homogenization. Furthermore, SLN can be prepared by precipitation both from microemulsions and emulsions containing organic solvents. The preparation of SLN with these methods involves several critical process parameters like high temperatures, pressure, high pressures, emulsifier concentration and toxicologically problematic solvents. The heat and cavitation due to high temperature & pressure cause significant thermodynamical and mechanical stress on the resulting product. In contrast, high emulsifier concentrations and residual solvents are more problematic for the application [21]. Ultrasonication method, a dispersing technique, is used for the production of solid lipid nanodispersion. It is simple, best and reproducible technique to get particle size in nano range (200-400nm) but it is reported that to obtain small particle size range (200-400nm) long sonication time (>15 min) is required which raises concerns about metal shed (metal toxicity) from the probe and contamination of the product [1].The modified solvent injection method is an another method for the preparation of SLN. This method is commonly employed for the preparation of liposomes and polymeric nanoparticles. The modified solvent injection method has several advantages over the existing methods such as the use of pharmaceutically acceptable organic solvents, no need for high pressure homogenization, no metallic contamination, easy handling and a fast production process without technically sophisticated equipments [22]. However, it has certain limitations like high PDI (polydispersity index) and non reproducibility of the method. In view of the several available methods and their attributes, it would be appropriate to select a suitable method for the preparation of SLN of RG using stearic acid as lipid. Therefore, it was proposed to prepare the SLN of RG by

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these two methods and characterize SLN for their particle size, zeta potential, entrapment efficiency, total drug content, morphology and in vitro drug release. 2. MATERIALS AND METHODS 2.1. Materials RG was a kind gift from Wockhardt Research Centre, Aurangabad, India. Stearic acid and Pluronic F68 and soya lecithin were generously supplied by SRL India. Dialysis membrane (molecular weight cutoff between 12000- 14000) was purchased from HiMedia, India. The other chemicals were of analytical reagent grade. 2.2. Preparation of SLN 2.2.1. Modified Solvent Injection Method RG-loaded SLN were prepared by a modified solvent injection technique [21]. Stearic acid (0.3% w/v) and soya lecithin (0.04% w/v) along with RG (0.02% w/v) were dissolved in 2 ml of dichloromethane and then immediately injected (1ml/min) through an injection needle (0.45 mm syringe diameter) into the 100 ml aqueous phase containing 0.25 gm of PL-F68 under continuous stirring (2000 rpm for 2 hrs) at room temperature. Then the solvent was evaporated in a vacuum oven at 40°C and pressure 200-400 mbar for 5 hrs. Blank (SLNR1) solid lipid nanoparticles were prepared without drug. 2.2.2. Ultrasonication Method Stearic acid (0.3% w/v) and soya lecithin (0.04% w/v) along with RG (0.02% w/v) were dissolved in 2 ml of dichloromethane and emulsified with 20 ml aqueous phase containing 1.25 gm of PL-F68, at 2000 rpm for 3 minutes and immediately intermittent ultrsonication (0.5 frequency ) was carried out for 15 min at 45% amplitude (Dr.Hielscher GmbH, 200H). After sonication the dispersion was diluted with 80 ml of distilled cold water under continuous stirring for 15 min. to obtain, a stable SLN suspension was obtained. Placebo (SLNR2) solid lipid nanoparticles were also prepared. The drug, lipid, surfactant concentration were kept constant, to observe the influences of the preparation method on the particle properties like particle size and zeta potential, entrapment efficiency, drug content, morphological study, and in vitro release studies. RG-SLN obtained using modified solvent injection and ultrasonication methods were abbreviated as RG-SLNR1 and RG-SLNR2, respectively. 2.3. Particle Size and Zeta Potential Analysis The particle size was measured by dynamic light scattering (Nano ZS, Malvern, UK) and zeta potential was estimated on the basis of electrophoretic mobility under an electric field. 2.4. Analytical Method HPLC analytical method was developed for determination of total drug content (TDC),entrapment

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efficiency (EE) and in vitro drug release of RG loaded solid lipid nanoparticles .The analytical method was a reversedphase HPLC (Cecil CE4201,India) in a binary mode, with a UV detector. The analysis was with a Phenomenex, C18 reverse phase, 250mm4.5 mm, 5 column maintained at 250C employing a mobile phase of acetonitrile (60%) and ammonium formate (0.01Mm) (40%) at pH 2.7 delivered at a flow-rate of 1ml/min. The calibration curve was linear in the concentration range of 10-100 g /ml (r2 = 0.9997) in Methanol. 2.4. Determination of Total Drug Content RG loaded SLN (1 ml) were diluted to 10 ml with chloroform/methanol mixture (1:2) and filtered through a 0.45 m filter paper .The total drug content was determined by HPLC (Cecil CE4201, India) method as described above. 2.5. Determination of Entrapment Efficiency SLN dispersion was centrifuged at 10,000 g for 30 minutes in a cooling centrifuge (-100C) .The supernatant was decanted and the sediment was washed with distilled water .The amount of RG in supernatant aqueous phase was estimated by HPLC and the entrapment efficiency was calculated by the equation: Entrapment efficiency (%) = Total drug content – Free dissolved drug X 100 Drug amount used 2.6. Determination of Repaglinide Solubility RG saturation solubility (Cs) was obtained by dispersing 300 mg drug in 100 ml of phosphate buffer pH 6.8 with 0.5% sodium lauryl sulphate (SLS). The suspension were stirred (100 rpm), at 37±0.5°C for 24 hrs (sufficient time for equilibration), filtered through a syringe filter (0.45m) and then assayed by HPLC (Cecil CE4201, India) method as described above.The experimental values were the average of three replicates (n=3). 2.7. In Vitro Release Study In vitro release studies were performed using dialysis bag diffusion technique [23]. Dialysis Membrane (molecular weight: 12,000 to14, 000) was soaked in double-distilled water for 12 h before use for experiment. 5 mg equivalent of repaglinide nanosuspension or repaglinide dispersion (repaglinide suspension in 0.5% aqueous sodium carboxymethylcellulose (NaCMC) were placed in the dialysis bag, sealed hermetically and immersed into 50 ml of phosphate buffer (pH 6.8) containing 0.5% SLS. The entire system was kept at 37±0.5°C with continuous magnetic stirring at 200 rpm. At fixed time intervals, the samples were withdrawn from receiver compartment; same dissolution media was replaced by fresh medium to maintain constant volume. Sink conditions were maintained for release studies (C1 0.05) in the zeta potential of nanoparticles was prepared by these two methods. Zeta potential depends on nature and amount of stabilizer used in the preparation of SLN. The surfactant and its amount were constant and this may be reason for insignificant difference in the zeta potential of the prepared SLN. The total drug content (assay of SLN formulations) in the nanosuspensions was around 98%. The EE of modified solvent injection and ultrasonication method were 75.14 ±1.29 %( RG-SLNR1) and 70.40 ±2.40 % (RG-SLNR2), respectively. The high EE was observed in both methods may be because of limited extraction of the drug towards the aqueous phase on account of low solubility of RG. However, there was significant difference (P>0.05) in EE of both methods. Modified solvent injection method has higher EE compared to ultrasonication method .The reason could be faster diffusion of drug towards outer phase because of smaller particle size in ultrasonication method. The smaller particle size lead to increased interface facilitating the drug diffusion towards the external aqueous phase. The saturation solubility (Cs) of RG in phosphate buffer pH 6.8 containing 0.5% SLS was 610.5 g/ml at 37±0.5°C. Sink conditions were maintained for release study: C1